Download Nap, a Novel Member of the Pentraxin Family, Promotes Neurite

The Journal
of Neuroscience,
April
15, 1996,
16(8):2483-2478
Nap, a Novel Member of the Pentraxin Family, Promotes Neurite
Outgrowth
and Is Dynamically
Regulated by Neuronal Activity
Cynthia C. Tsui,’ Neal G. Copeland,
and Paul F. Worleyls*
Debra
J. Gilbert,4
Nancy
A. Jenkins,4
Carol
Barnes,3
Departments of 1Neuroscience and *Neurology, The Johns Hopkins University School of Medicine, Baltimore, Maryland
21205, 3Department of Psychology, Neurology, and Division of Neuronal Systems, Memory, and Aging, University of
Arizona, Tucson, Arizona 84724, and 4Mammalian Genetic Laboratory, ABL-Basic Research Program, NC/-Frederick
Cancer Research and Development Center, Frederick, Maryland 2 1702
Stimulus-linked
RNA and protein synthesis is required for establishment of long-term neuroplasticity.
To identify molecular
mechanisms
underlying
long-term
neuroplasticity,
we have
used differential cDNA techniques to clone a novel immediateearly gene (IEG) that is rapidly induced
in neurons of the
hippocampus
and cortex by physiological
synaptic activity.
Analysis of the deduced amino acid sequence indicates homology to members of the pentraxin family of secreted lectins that
include C-reactive protein and serum amyloid P component.
Regions of homology include an 8 amino acid “pentraxin signature” sequence
and a characteristic
pentraxin
calciumbinding domain. We have termed this gene and the encoded
protein Narp (from neuronal activity-regulated
pentraxin). Biochemical analyses confirm the presence of a functional signal
sequence, and Narp is secreted by transfected
COS-1 cells in
culture. Additionally,
Narp binds to agar matrix in a calciumdependent
manner consistent with the lectin properties of the
pentraxin family. When cocultured with Narp-secreting
COS-1
cells, neurons of cortical explants exhibit enhanced growth of
neuronal dendritic processes. Neurite outgrowth-promoting
activity is also observed using partially purified Narp and can be
specifically immunodepleted,
demonstrating
that Narp is the
active principle. Narp is fully active at a concentration
of -40
rig/ml, indicating a potency similar to known peptide growth
factors. Because Narp is rapidly regulated by neuronal activity,
its lectin and growth-promoting
activities are likely to play role
in the modification of cellular properties that underlie long-term
plasticity.
The mature CNS exhibits the capacity to alter cellular interactions
as a function of the activity of specific neuronal circuits. This
capacity is believed to underlie learning and memory as well as
aspects of postnatal development
of the brain (Shatz, 1990).
Cellular mechanisms underlying activity-dependent
plasticity are
known to be initiated by rapid, transmitter-induced
changes in
membrane conductance properties and activation of intracellular
signaling pathways (Bliss and Collingridge,
1993). Several lines of
evidence also indicate a role for rapid synthesis of mRNA and
protein in long-term neuroplasticity. For example, classical studies
of learning and memory demonstrate a requirement
for protein
synthesis in long-term, but not short-term, memory (Flexner et al.,
1963; Agranoff, 1981; Davis and Squire, 1984) and long-term
enhancement of synaptic connectivity, studied in cultured invertebrate neurons (Montarolo
et al., 1986; Bailey et al., 1992) or in
the rodent hippocampus (Frey et al., 1993; Nguyen et al., 1994) is
blocked by inhibitors of either RNA or protein synthesis. Impor-
tantly, inhibitors of macromolecular
synthesis are most effective
when administered
during a brief time window surrounding
the
conditioning
stimulus, indicating a special requirement for molecules that are rapidly induced (Goelet et al., 1986).
Immediate-early
genes (IEGs) are rapidly induced in neurons
by neurotransmitter
stimulation
and synaptic activity and are
hypothesized to be part of the macromolecular
response required
for long-term plasticity (Goelet et al., 1986; Sheng and Greenberg,
1990; Silva and Giese, 1994). To identify cellular mechanisms that
may contribute to long-term plasticity in the vertebrate brain, we
and others have used differential cloning techniques to identify
genes that are rapidly induced by depolarizing
stimuli (Nedivi et
al., 1993; Qian et al., 1993; Yamagata et al., 1993, 1994a,b;
Andreasson and Worley, 1995; Lyford et al., 1995). In contrast to
the earlier focus on transcription
factors, many of the newly
characterized IEGs represent molecules that can directly modify
the function of cells and include growth factors (Nedivi et al.,
1993; Andreasson and Worley, 1995) secreted enzymes that can
modify the extracellular matrix, such as tissue plasminogen activator (Qian et al., 1993), enzymes involved in intracellular signaling, such as prostaglandin synthase (Kaufmann et al., 1996; Yamagata et al., 1993) and a novel homolog of H-Ras, termed Rheb
(Yamagata
et al., 1994b), as well as a novel cytoskeletonassociated protein, termed Arc (Lyford et al., 1995). The remarkable functional diversity of this set of rapid response genes is
representative
of the repertoire of cellular mechanisms that are
likely to contribute to activity-dependent
neuronal plasticity.
Here we describe a novel IEG that is homologous
to the
Received July 3, 1995; revised Jan. 19, 1996; accepted Jan. 24, 1996.
This work was supported by National Institutes of Health Grants MHS3608
(P.F.W.), AGO9219 (C.A.B., P.F.W.), a grant from the W. M. Keck Foundation, the
DeVelbliss Fund, and the Krieger Mind/Brain
Institute (P.F.W.), and by the National Cancer Institute, Department of Health and Human Services, under contract
N01-CO-74101
with ABL. We thank Anthonv Lanahan for help in construction of
cDNA libraries and Gopal Thinakaran, Maha Papapavlou, and D. Barnhart for
excellent technical assistance. We also thank Daniel Nathans for support in the initial
cloning of Narp.
Corresoondence should be addressed to Paul F. Worley, Departments of Neuroscience and Neurology, The Johns Hopkins University School of Medicine, 725
North Wolfe Street, Baltimore, MD 21205.
Copyright 0 1996 Society for Neuroscience
0270-6474/96/162463-16$05.00/O
Key words: growth factors; immediate-early genes; lectins;
long-term potentiation; neurite outgrowth: pentraxins
2464
J. Neurosci.,
April
15, 1996,
76(8):2463-2478
pentraxin family of secreted, calcium-dependent
lectins, which
includes C-reactive protein (CRP) and serum amyloid P component (SAP). We have termed this gene and the encoded protein
Nulp (from neuronal activity-regulated
pentraxin).
Our studies
demonstrate that Nurp is a secreted protein with biochemical
properties of a calcium-dependent
lectin. NUT mRNA is abundantly expressed in neurons of the developing and adult brain and
spinal cord and is rapidly regulated by physiological
synaptic
activity. Functional studies indicate that Narp promotes neuronal
migration and dendritic neurite outgrowth of neurons using cortical explant cultures, and it does so with a potency that is
comparable to known neurotrophins
and growth factors. Because
Narp is rapidly regulated by physiological synaptic activity, our
observations suggest a role for Nurp in neural development and
activity-dependent
neuronal plasticity.
MATERIALS AND METHODS
Animals und supplies. Adult male Sprague-Dawley
or Fischer-344
[longterm potentiation
(LTP)
studies]
rats were used in studies of Nurp
regulation.
Developmental
studies used male and female Sprague-Dawley pups of the indicated
age. Radiochemicals
were obtained
from DuPont NEN (Boston, NEN). AlI other reagents were from Fisher (Orangcburg. NY) and Sigma (St. Louis. MO) unless snecificallv
noted.
&nst&ion
anYd scrkening of the sudtructed ci)NA lihr&y. A subtracted
cDNA library was constructed
as described
previously
(Yamagata
ct al.,
1993). The subtracted
library was screened with [“P]cDNA
prepared
by
reverse transcription
of poly(A+)
RNA prepared
from hippocampus
of
control
or seizure-stimulated
rats pretreated
with cycloheximide
(20
mg/kg, i.p.) as described
previously
(Yamagata
et al., 1993). Near fulllength cDNAs
of rat Nurp were isolated
by iterative
screening
of an
unsubtracted,
oligo(dT)-primed
cDNA library prepared
from hippocampus 4 hr after a maximal
electroconvulsive
seizure (MECS).
DNA sequencing. Both strands of three independent,
near full-length
Nurp cDNAs were sequenced as double-stranded
plasmids with synthetic,
specific primers
using the dideoxynucleotide
chain termination
method
with deoxyadenosine
5’-[a-“Slthiotriphosphate
and Sequenase
(United
States Biochemicals,
Cleveland,
OH).
Northern
analysis. This procedure
was performed
as described
previously (Linzer and Nathans, 1983) with 10 pg of total RNA per lane. RNA
was isolated by standard CsCl density centrifugation
and assayed for yield
and purity by ultraviolet
spectroscopy.
Gels were stained with ethidium
bromide,
and the ribosomal
bands were visualized
to assess equal loadings of RNA. The probe used for Northern
analysis was a 1.8 kb 3’-end
fragment
of Nurp cDNA. The cDNA fragment
was labeled by the random
priming technique
(Pharmacia,
Uppsala,
Sweden) using [olla’P]dCTP.
In situ hvbridizution.
Freshlv dissected brain tissue was raoidlv frozen in
plastic molds placed on a dry ice/ethanol
slurry as described previously
(Cole et al., 1990). Control
and experimental
tissues were frozen in the
same tissue block to ensure identical
conditions
during tissue sectioning,
subsequent
storage, and in situhybridization.
“S-labeled
Nurp antisense
riboprobe
was prepared
from an appropriately
restricted
pBluescript
plasmid containing
the near full-length
cDNA. In situ hybridization
was
performed
as described
previously
(Saffen et al., 1988). Selected slides
were treated with photographic
emulsion (Kodak
NTB2, Rochester,
NY)
and counterstained
as previously
described
(Jordan,
1990). Semiquantitative analysis of autoradiographic
images was performed
using a computerized
densitometer
(Loats Associates,
Westminster,
MD).
Electrophysiology.
Seizures were induced in adult male Sprague-Dawley rats by MECS using a constant-current
signal generator
(ECT unit,
Ugo, Basil, Switzerland)
as described
previously
(Cole et al., 1990). For
LTP studies, Fischer-344
rats were implanted
bilaterally
with stimulating
and recording
electrodes
in the perforant
path and hilus of the dentate
gyrus as described previously
(Worley
et al., 1993). Rats were allowed to
recover
for at least 2 weeks before
any recordings
were performed.
Twelve chronically
implanted
rats received
high-frequency
(HF) stimulation in one hemisphere
and low-frequency
(LF) stimulation
in the other
hemisphere.
Electrical
stimuli consisted of 200 msec diphasic, constantcurrent
pulses given at a stimulus
intensity
of 500 FA. The LF test
stimulation
was delivered
at 0.1 Hz, and the HF stimulation
parameters
consisted of 50 repetitions
of a 20 msec train (i.e., 8 pulses) delivered
at
400 Hz (400 total pulses). All response data were digitized
by computer
at 20 kHz and stored on disk for subsequent
off-line analysis. The field
Tsui et al.
l
Nafp
Expression
and
Regulation
in Brain
EPSP amplitude
was measured
as the voltage difference
between
two
cursors set at the EPSP onset and 1 msec later. The HF parameters
reliably induce LTP (Worley
et al., 1993) and increases in EPSP amplitude, assayed 25 min after the stimulus, ranged from 20 to 30%. After this
treatment,
the rats were killed at 30 min (n = 6), 1 hr (n = 2) 2 hr (n =
2) 4 hr (n = 2) or 24 hr (n = 2).
Three additional
animals were pretreated
with MK-801
(1 mgikg, i.p.)
1 hr before the delivery of the HF stimulus. The intensity of the stimulus
was adjusted such that the postsynaptic
population
response was identical
to that before
MK-801
administration.
The MK-801
administration
blocked
LTP. Animals
were killed 2 hr after the HF stimulus
and
processed
for in situ hybridization.
Monocular
deprivation.
Monocular
deprivation
was performed
in adult
Sprague-Dawley
rats as described
previously
(Worley
et al., 1991) usingan intravitreal
injection
of the sodium channel antagonist
tetrodotoxin
(TTX; 10 kl of 200 &M solution of TTX in PBS). To assess the effectiveness of TT’X injections,
its anticipated
blockade
of the consensual
pupillary light reflex was monitored
immediately
after injection
and before
killing.
Identical
treatment
in the absence of TTX was performed
in
control rats.
In vitro transcription
and translation.
Nurp protein
was synthesized
using in vitro transcription
and translation
(TNT) in a coupled reticulocyte lysate system (Promega,
Madison,
WI). Template
DNA (I pg) was
used with rabbit reticulocyte
lysate, TNT reaction buffer, amino acid mix
(1 mM) minus methionine,
[3”S]methionine
(1000 Ciimmol)
at 10 mCi/ml,
RNasin
ribonuclease
inhibitor
(40 U/ml),
and T3 RNA polymerase
(2
U/ml).
&$I and &-zI were used to linearize
the DNA plasmids.
TNT
reaction products were analyzed by SDS-PAGE.
The polyacrylamide
gels
were first fixed in 10% (w/v) trichloroacctic
acid, 10% (v/v) glacial acetic
acid, and 30% (v/v) methanol
followed
by soaking in autoradiography
enhancer
(Fluoro-Hance,
Research Products International,
Mt. Prospect,
IL) before the gels were dried and exposed to autoradiography.
Analysis ofpost-trunslutionul
modification
of Narp. TNT reactions were
performed
as described
above except with the addition
of 2.5 pg of dog
microsomes
(Promega).
For the microsomal
proteolytic
protection
assays, circular full-length
Nurp plasmid DNA (1 pg) or 5’-truncated
Nurp
plasmid DNA (1 Fg) was used as template
to synthesize full-length
Narp
protein
(in the presence or absence of microsomes)
or a 5’-truncated
Nurp lacking the first 46 amino acids, respectively.
Proteins were exposed
to proteolytic
digestion
by trypsin
(0.1 mg/ml), with or without
Triton
X-100 (0.1%).
Reactions
were analyzed
by SDS-PAGE.
Deglycosylation
of Nurp was performed
by first adding SDS (0.5%)
to the reticulocyte
lysate and boiling for 2 min to lyse the microsomes.
Glycosidase
reactions
were carried
out at 37°C overnight
with endoglycosidase
H (6 mu;
Boehringer
Mannheim,
Indianapolis,
IN) in reaction
buffer (150 ItIM
sodium acetate, pH 5.5).
Generation
of Narp polyclonal
antisera. The full-length
Nurp cDNA
sequence, exclusive of the putative
signal sequence, was generated
using
PCR primers
that encoded
flanking
restriction
enzime
sequences
(EcoRI,
5’-primer;
BumHI,
3’-primer).
The amnhfied
Naro insert was
subcloned in frame into pTrcHisA
prokaryotic
expression
vector containing an N-terminal
hexahistidine
(Invitrogen,
San Diego, CA). Full-length
Nurp protein was isolated and purified using the Niz+ agarose purification
system (Qiagen,
Hilden,
Germany).
Eluted fractions
were separated
on
preparative
SDS-PAGE,
and the specific band was excised from the gel.
Approximately
1 mg of the recombinant
Nurp protein
was used to
immunize
each rabbit (HRP, Denver, PA). The crude antisera specifically
recognized
the bacterial
recombinant
protein
and the in vitro TNT
product.
Metabolic
labeling of COS-I
cells und immunoprecipitation
of’ Narp.
Full-length
Nurr, cDNA
insert was subcloned
into a CMV-uromoter
I
containing
mammalian
expression
construct
(pRK5;
Genentech,
San
Francisco,
CA). COS-1 cells were transiently
transfected
with 10 Fg of
plasmid
DNA
(vector
alone and Nurp-expressing
construct)
using the
calcium phosphate
method (Chen and Okayamam,
1987). For metabolic
labeling
of COS-1 cells, 250 mCi/ml
[truns-35S]methionine
(ICN Biochemicals,
Costa Mesa, CA) was added with a media change 40 hr after
the initial transfection
in DMEM
(Gibco,
Gaithersburg,
MD)
lacking
methionine
and supplemented
with 1% fetal bovine serum (HyClone,
Logan, UT) for 3 hr. Conditioned
media were analyzed
by SDS-PAGE
and autoradiography.
Immunoprecipitation
of Nurp from the COS-1 cell conditioned
media
was performed
as follows. Conditioned
media were first cleared of cellular debris by centrifugation
for 15 min at 1500 X g. Samples were
adjusted to 1X immunoprecipitation
buffer (150 mM NaCI, 50 mM Tris_
a
,
Tsui et al. . Narp Expression
and
Regulation
in Brain
HCl, pH 7.4, 0.5% NP-40, 0.5% sodium deoxycholate,
5 mM EDTA, 50
Fg/rnl pepstatin,
50 pg/ml leupeptin,
10 &ml
aprotinin,
and 0.25 mM
phenylmethylsulfonyl
fluoride)
and precleared
by mixing with 50 ~1 of
protein A agarose (Pierce, Rockford,
IL) for 30 min at 4°C (Sisodia et al.,
1990). Secreted Nurp was immunoprecipitated
by mixing 3 ~1 of polyclonal Nurp antisera with the conditioned
media at 4°C for 6 hr. The
immune complexes were collected
by adding 50 ~1 of protein A agarose
(Pierce).
Samules were resusoended
in SDS eel loading buffer. boiled for
5 min before ‘SDS-PAGE,
and analyzed
by &toradio&aphy.
Calcium-dependent
binding of Narp to agar and aj2ypurification.
Narp
was harvested
from conditioned
media of transiently
transfected
COS-1
cells as described
above. For initial
studies examining
the substratebinding properties
of Nurp, columns
(0.5 ml of gel matrix)
were prepared
with Sepharose-phosphatidylcholine
(Pharmacia),
Sepharosephosphatidylethanolamine
(Sigma),
pulverized
4% agarose (Sigma),
or
pulverized
4% agar (Sigma).
Columns
were washed
in a calciumchelating elution buffer (0.15 M NaCl, 20 ITIM EDTA, 0.05 M Tris, pH 7.4)
and equilibrated
in Narp-binding
buffer (0.15 M NaCl, 10 mM CaCl,, 0.05
M Tris, pH 7.4). Conditioned
media from ten 10 cm plates of COS-1 cells
transfected
with either the Nurp expression
vector or the same vector
without
insert were applied
to the columns
and were washed with 2
column volumes of the binding buffer. Proteins that bound to the column
were eluted in calcium-chelating
elution buffer. Proteins
that remained
adherent ,to the columns were solubilized
in SDS-loading
buffer [6% SDS,
15% (v/v) P-mercaptoethanol,
30% (v/v) glycerol, 0.350 M Tris, pH 6.81.
All
fractions
were
analyzed
by SDS-PAGE
and visualized
by
autoradiography.
In experiments
that examined
the neurite outgrowth-promoting
effects
of Nurp, we used agar column chromatography
to partially purify Nurp or
myc-tagged
Narp. The c-myc epitope
tag was subcloned
into the C
terminus
of Narp-pRK5
construct.
c-myc epitope
(MEQKLISEEDLN)
was designed with a 5’-EcoRI
and a 3’-BamHI
restriction
sequence site.
Two 27-mer PCR primers
containing
flanking
Nurp sequence were designed with an EcoRI site in the 5’-PCR primer and a BarnHI
site in the
3’-PCR primer along with a stop codon. A total of 100 ml of COS-1 cell
conditioned
media was incubated
with 20 ml of pulverized
agar suspension in binding
buffer (same as above)
at 4°C for 4 hr followed
by
subsequent
washes with binding
buffer. Nurp was eluted in four 1 ml
fractions
with elution buffer. The yield of myc-tagged
Nurp was determined by direct competitive
ELISA
(described
below) and was typically
-250 ng/lO cm dish.
Microexplant
cultures of the cerebral cortex. Cortices were dissected from
postnatal
day 1 (Pl) rat pups and dissociated
into cortical
explants by
passing the tissue through a 24-gauge needle. Explants were washed twice
with minimum
essential
medium
(MEM;
Gibco)
supplemented
with
glucose (6 gm/l) by sedimentation
and resuspension
in fresh media. The
explants were then plated in 35 mm tissue culture
dishes coated with
poly+ornithine
(Sigma). The tissue culture dishes were coated with 1 ml
of poly-L-ornithine
(0.1 mg/ml) overnight
at room temperature
and rinsed
twice with d-H,0
before culturing
explants in MEM
with -7-8
explants
per dish in a 5% CO,-humidified
incubator
at 37°C.
Transiently
transfected
COS-1 cells (with pRK5 vector alone, pRK5
containing
the full-length
Nurp, or pRK containing
a C-terminal
myctagged Nurp construct)
were trypsinized
and harvested
24 hr after transfection.
Aggregates
of COS-1
cells (5 X 10’ cells) were formed
by
inverted hanging drop cell culture (Kennedy
et al., 1994) for 16-18 hr and
transferred
to cortical explant cultures. A single aggregate
was added to
each 35 mm dish. Cocultures
were maintained
for 24 or 48 hr, and neurite
outgrowth
from each explant was assessed by light microscopy
(magnification 150x) and scored in triplicate
dishes by an observer
blind to the
protocol.
The criteria
for scoring an explant
as “positive”
for neurite
outgrowth
were the presence of neurites that surrounded
the periphery
of
the explant and extended
radially over the substrate
at least lo-15
cell
body lengths. In other control experiments,
we examined
the effect of
coculturing
cortical explants with COS-1 cells expressing
amyloid precursor protein-like
protein
(Slunt et al., 1994) from the mammalian
expression vector CB6.
In other experiments,
myc-tagged
Narp was partially
purified
from
COS-1 cell supernatant
using agar column chromatography
as described
above and examined
for neurite outgrowth
activity. The concentration
of
myc-Narp
was quantitated
using a direct competitive
ELISA
described
below. Differing
concentrations
of myc-Narp
were added to culture
dishes, and the effects on neurite outgrowth
were examined.
Immunodepletion
of myc-tagged
Nurp was performed
by incubating
partially purified myc-tagged
Nurp prepared
from COS-1 cell-conditioned
J. Neurosci.,
April
15, 1996,
76(8):2463-2478
2465
media (-60
ng of Nurp)
with mouse anti-myc
monoclonal
antibody
(mAb; 0.5 pg, Oncogene
Research Products, Cambridge,
MD) previously
conjugated
with G-protein-agarose
beads. The anti-myc-G-protein
conjugate was prepared
by incubating
the Ab with 1 ml of gel slurry, (Pierce)
for 2 hr in IgG-binding
buffer (0.1 M sodium acetate, pH 5.0) at 4°C
followed
by centrifugation.
The anti-myc
G-protein
conjugate
was then
resuspended
in buffer (0.15 M NaCl, 0.05 M Tris, pH 7.4) containing
Nurp
and incubated
overnight
at 4°C. Control
experiments
substituted
mouse
fluid ascites (0.5 pg, Sigma) for the anti-myc
Ab. The control immunodepletion
experiment
procedure
was identical
to the anti-myc
Ab
experiment.
Quantitation
of myc-tagged
Narp using direct competitive
ELISA.
myctagged Narp was quantitated
using a standard
antigen-inhibition
curve
generated
with serial dilutions
of a standard c-myc peptide (amino acids
412-418,
Santa Cruz Biotechnology,
Tebu, France).
Partially
purified
myc-tagged
Nurp was coated onto a 96-well, flat-bottom,
high-binding
plate (EWRIA,
Costar, Cambridge,
MA) for 1 hr at room temnerature.
hells‘were
rinsed three times with wash buffer (0.5% Surf&t-Amps
Tween 20,1% Blocker BSA in PBS, and 1 pack of BupH Dulbecco’s
PBS,
Pierce)
and blocked
with blocking
buffer (10% Blocker
BSA in PBS,
Pierce) for 30 min at room temperature.
Primary
mouse anti-myc
Ab at
1000 rig/ml dilution
(Oncogene
Research
Products)
was preincubated
with known
amounts
of c-myc peptide
for 1 hr at room temperature
before applying
to wells coated with partially
purified
myc-tagged
Nurp.
After 2 hr incubation
at room temperature,
the wells were rinsed three
times with wash buffer, and goat anti-mouse
IgG peroxidase-conjugated
Ab (l:lOOO,
Pierce) was added for I hr at room temperature.
2,2’Azinobis(3-ethylbenzothiazoline)-6
sulfonic
acid diammonium
salt reagent (ABTS; Pierce) was used as the peroxidase
substrate solution,
and
the hydrolysis
was measured
using an ELISA reader with a 410 nm filter.
Concentration
of Nurp was determined
by calculating
the amount
of
c-myc peptide necessary to competitively
inhibit half-maximal
binding of
anti-myc
Ab to myc-Nurp.
Immunohistochemistry.
Cortical
explants
cocultured
with COS-1 cells
from Pl rat pups were prepared
as described above. Cultures were rinsed
with HBSS (Gibco)
at 37°C air-dried
for 20 min, and fixed with 3.7%
formaldehyde
in 10 mM PBS, pH 7.4, for 30 min at room temperature.
Explants were blocked and permeabilized
with a solution containing
0.5%
Triton X-100, 10% normal blocking serum (Vector
Laboratories.
Burlingame, CA) in PBS for 4 hr at 4°C and then incubated
in primary
Abs
[anti-microtubule-associated
protein-2
mAb (MAP-2)
at 1:lOOO (SMI 52,
Sternberger
Monoclonals
Incorporated),
anti-tau
mAb at 1:200 (Boehringer Mannheim),
anti-glial
fibrillary
acidic protein
mAb (GFAP)
at
1:400 (Chemicon,
Temecula,
CA), or control mouse ascites fluid at 1:200
(Sigma)]
overnight
at 4°C. After rinsing off the primary
Abs, endogenous
peroxidase
was quenched
by incubating
with 1% H,O, in PBS for 15 min
at room temperature.
Cultures
were incubated
further with biotinylated
anti-mouse
secondary
Ab (50 Fliml, Vector)
in PBS for 1 hr at room
temperature,
and immunoreactivity
was visualized
using the Vectastain
Elite ABC and DAB substrate
(Vector
Laboratories,
Burlingame,
CA).
Interspecific
mouse backcross mapping. Interspecific
backcross progeny
were generated
by mating
(C57BU6J
X M. spretus) F, females and
C57BL/6J
males as described previously
(Copeland
and Jenkins, 1991). A
total of 205 N, mice were used to map the Nurp locus. DNA isolation,
restriction
enzyme digestion,
agarose gel electrophoresis,
Southern
blot
transfer,
and hybridization
were performed
as described previously
(Jenkins et al., 1982). All blots were prepared
with Hybond-N+
nylon membrane (Amersham,
Arlington
Heights,
IL). The probe,
an -500
bp
EcoRIiBamHI
fragment
of rat cDNA corresponding
to the 3’-end of the
open reading frame (ORF),
was labeled with [a-32P]dCTP
using a nick
translation
labeling kit (Boehringer
Mannheim);
washing was done to a
final stringency
of 0.8X SSCP, 0.1% SDS, 65°C. A major fragment
of 6.4
kb was detected in SphI-digested
C57BL/6J
DNA, and a fragment
of 7.6
kb was detected
in SphI-digested
M. spretus DNA.
The presence
or
absence of the 7.6 kb M. spretus-specific
5phI fragment
was followed
in
backcross
mice.
A description
of the probes and RFLPs
for the loci linked to Narp
including
erythropoitin
(Epo)
and platelet-derived
growth
factor-a
(Pdgfu) has been reported
previously
(Singh et al., 1991). One locus has
not been reported
previously
for our interspecific
backcross:
FMS-like
tyrosine
kinase 3 (Flt3). The probe was an -850 bp fragment
of mouse
cDNA that was kindly provided
by Ihor Lemischka
(Princeton
University,
Princeton,
NJ). The probe detected 11.5,7.5, and 4.3 kb and 11.5,7.5,6.0,
and -1.0 kb QhI fragments.
The presence or absence of the 6.0 and -1.0
kb M. spretus-specific
.SphI fragments,
which cosegregated,
was followed
2466
J. Neurosci.,
April
15, 1996,
76(8):2463-2478
in backcross mice. Recombination distances were calculated as described
previously (Green, 1981) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.
Sequence analysis. cDNA and amino acid sequences were analyzed
using Geneworks (IntelliGenetics, Mountain View, CA), Strider (Commissariat al’Energie Atomique, Gif-Sur-Yvette Cedex, France), ProteinPredict (Rost and Sander, 1993, 1994), and SBASE (Pongor et al., 1994).
RESULTS
Narp cDNA sequence
Anovel cDNA corresponding
to the 3’-noncoding
region of an
-2.5 kb mRNA was identified by differential
screening of a
subtracted cDNA library prepared from seizure-stimulated
hippocampus (Yamagata et al., 1993). A near full-length cDNA was
identified by iterative screening of an unsubtracted cDNA library
prepared from hippocampus. The size of the cDNA (2561) corresponds closely to the estimated size of the mRNA determined
by Northern analysis. The longest ORF with an initiator methionine predicts a 432 amino acid protein. A second, independent
clone (2460 bp that begins at nucleotide 101 of the longest clone)
contains the entire ORF, and the nucleotide and the predicted
amino acid sequences are identical to those of the longer clone.
Figure 1 shows the complete cDNA and deduced protein sequences. The initiator methionine was selected to be the best
Kozak consensus for translation initiation and occurs at nucleotide 128 of the longest cDNA (Kozak, 1987). The ORF continues
to the 5’-end of the cDNA, and there is no in-frame stop 5’ to the
predicted initiator methionine. Alternative initiator methionines
are present at nucleotides
265, 271, and 466. The identified
location of the translation start site is supported by analysis of the
in vitro transcription/translation
products (see below). The 3’noncoding region contains the ATTTA
motif in positions 2362
and 2396, which may contribute to the rapid turnover of Narp
mRNA (Shaw and Kamen, 1986) and is present in many IEGs. No
classical polyadenylation signal sequence (AATAAA)
was identified; however, the sequence ATTAAA
is present in 5 independent
clones 21 nucleotides from the 3’-terminal poly(A) tail.
Predicted protein sequence of Narp: homology to the
pentraxin family
Nurp has a calculated molecular weight of 46.2 kDa and a p1 of
5.46. There is an N-terminal secretory signal peptide sequence
(underlined in Fig. 1) predicted using methods from von Heigne
(1986). The predicted cleavage site is between glycine and glutamine. Three potential N-linked glycosylation sites were identified (Geneworks program) at amino acids 133, 174, and 378.
Nurp is 24 and 26% identical to CRP and SAP, respectively,
over a 210 amino acid span that includes the full-length sequence
of mature CRP and SAP and the C-terminal 216 amino acids of
NUT (Dowton and McGrew, 1990; Rassouli et al., 1992). Figure
2A shows the alignment of Narp with rat CRP and SAP. Islands of
homology are clustered throughout
the sequence. Eight amino
acids have been identified that constitute the “pentraxin
family
signature” sequence (H-X-C-X-S/T-W-X-S/T),
which is present in
all pentraxin family members (Breviaro et al., 1992) and is also
present in Narp.
The recent resolution of the crystal structure of SAP provides
detailed structure-function
information for the pentraxin family
and identifies several functional domains (Emsley et al., 1994). As
isolated from ascites or pleural effusion fluids, SAP is a decameric
molecule composed of identical subunits noncovalently associated
in two pentameric rings that interact face to face. Each of the
Tsui et al.
l
Narp
Expression
and
Regulation
in Brain
monomers contains 15 antiparallel P-strands (designated strands
A-O, Fig. 2A) that form two p sheets. Corresponding
regions of
NUT are predicted to form P-strands as predicted by computer
modeling (Rost and Sander, 1993, 1994). The pentraxin signature
sequence corresponds
to strand H. The region of Narp with
highest homology to SAPICRP spans the sequence between
P-sheets K and L. This region of SAP is important in binding
calcium (Emsley et al., 1994). SAP binds two calcium ions with
different affinities. The higher-affinity binding site includes coordinating side chains of amino acids Asp-58, Asn-59, Glu-136,
Asp-138, and the main-chain carbonyl of Gln-137. All of these
residues are present at homologous positions in Narp, with the
single exception that Narp encodes an Ala at homologous position
58 (Fig. 2A). Asp-58 is present in hamster SAP, human CRP, and
Limulus CRP but varies in CRPs from other species (Emsley et
al., 1994). The second calcium ion is coordinated
by Glu-136,
Asp-138, and Gln-148. Each of these residues is conserved in
Narp. SAP binding to derivatized sugars involves the amide nitrogens of Gln-148 and Asn-59, which are oriented in the proper
position to form hydrogen bonds with the sugar derivative via
interactions of their amide oxygens with the calcium ions (Emsley
et al., 1994).
Cysteine residues Cys-36 and Cys-95 form a disulfide link between adjacent strands C and L and are present in most members
of the pentraxin family (Emsley et al., 1994). Cysteines are present
in the homologous positions in Nary. Based on these homologies,
we conclude that Nurp is a member of the pentraxin family.
Computer modeling of the N-terminal 200 amino acids of Narp
predicts that this region possesses a high degree (61%) of
a-helical secondary structure (Rost and Sander, 1993, 1994).
Helical wheel analysis (Cohen and Parry, 1994) of two of these
putative helical domains (amino acids 40-82 and 105-132) indicates that they are strongly amphipathic with heptad repeats of
hydrophobic amino acids (data not shown).
Four additional novel pentraxins have been reported recently
that are similar to Narp in that they exhibit the pentraxin consensus sequences in the C-terminal half of the molecule but possess
an extended N-terminal
sequence that is novel. Information regarding the biochemistry and function of these molecules is limited and is based primarily on their sequence homology to SAP
and CRP. TSG-14 is rapidly induced in endothelial cells by growth
factor stimulation and is hypothesized to modify the extracellular
matrix of the endothelium (Breviaro et al., 1992; Lee et al., 1993).
Overall, TSG-14 is 22% identical to Nurp but is similar to Narp
to the penin that it encodes -200 amino acids N-terminal
traxin homology region, and this domain is predicted to possess
a high degree of a-helical structure (Rost and Sander, 1993,
1994).
Another pentraxin, termed NP (from neuronal pentraxin), was
purified from brain membranes as a binding protein for the snake
venom toxin taipoxin (Schlimgen et al., 1995). NP is similar in size
to Narp and TSG-14, and its amino acid sequence is 45% identical
to Narp. NP is expressed in discrete populations of neurons in the
hippocampus, cortex, and brainstem, indicating a partially overlapping pattern of expression with Nurp. The regulation of NP,
particularly whether it is an IEG, has not been examined. NP
potentiates the toxicity of taipoxin on glia and is hypothesized to
play a role in reuptake of extracellular proteins. The third novel
pentraxin, termed Apexin, was purified and cloned independently
by two groups from guinea pig sperm (Noland et al., 1994; Reid
and Blobel, 1994). The Apexin amino acid sequence is 90%
identical to Narp and, therefore, Apexin may represent the guinea
Tsui et al.
l
Nap
Expression
and
Regulation
in Brain
J. Neurosci.,
April
15, 1996,
TGGTGCTGGCGTTTCCCTGCTTGCACGCGGTTCCCTCGAGCGCCGCTC
CGACCCACGTAGCCGGCCGCCAAGGCGCCCAGACGGCAACGCGAG
ATG CTG GCG CTG CTG ACC GCC GGC GTG GCG CTC GCC GTG GCC GCG GGA CAA GCC CAG GAT
-16
M
MC
L
A
CCG ATA
L
L
T
A
G
CCT GGC AGT CGC TTC
V
A
L
GTG TGC ACC
A
V
A
A
G
GCG CTG CCC CCC GAA
ij
A
Q
76(8):2463-2478
2467
48
127
206
D
GCG GCG CGC GCC
266
CCT GAG GAG GAG CTG
326
5NPIPGSRFVCTALPPEAARA
CCC TGC
25G
C
CCG CTG
P
L
CCC GCG ATG CCC ATG CAG GGA GGC GCG CTG AGC
P
A
M
P
M
Q
G
G
A
L
S
P
CGA GCC GCT GTG CTG CAC TGG CGC GAG ACC GTC GTG CAG CAG AAG
45R
A
A
V
L
CAG CGA GAA GCC ATC
65Q
R
E
A
I
H
W
CGA GM
R
GCT AAG GCG CGC GGC ACG
E
R
E
T
V
V
Q
Q
K
T
S
X
L
A
R
E
E
GAG ACG CTG
E
CTC ACC AGC AAG CTG GCC CCC TGI
L
E
T
L
E
G
GGC GCT
G
GAG GGA Cl-A
C
L
L
CCC GGC
A
GGG GCC ACG GGC AAG GAC ACC ATG GGC GAC CTG CCG
386
A
446
G
CGG GAC
506
GAC CGC TTG GAG
566
85GKARGTGATGKDTMGDLPRD
CCG GGC CAC GTC GTG GAG CAG CTT AGC CGC TCG CTG CAG ACC CTC AAG
105
P
AGC
125
S
G
E
L
V
CTG GM
165
185
205
L
E
MC
ACA
N
T
MG
TCA
K
S
E
I
K
GCC TCG
245
A
L
S
V
L
L
Q
D
E
CTG MT
L
N
CCA GAT
P
K
D
L
P
T
D
G
CCC TGG
325
P
W
P
I
G
G
G
Q
L
H
R
L
E
L
F
M
W
S
A
L
GCA TTC
A
L
I
W
F
D
R
P
G
N
H
P
R
F
V
L
I
I
CCT GTG GAG ACG
405
P
V
R
S
GCG TCT
L
Q
MT
T
L
G
E
L
GM
E
I
E
T
L
Q
AAA GI'G
K
E
G
T
V
L
P
GGT AAC MT
G
S
N
R
GCA TTC
A
K
F
A
R
A
S
Y
N
F
V
L
G
TGC
C
W
R
Q
GAG ACC TCG
T
P
GCC TTC
A
S
CCC ATA
P
K
L
Q
R
K
E
L
E
GGC MC
R
G
F
I
Y
L
R
ACC ATC TGC
T
I
T
Q
V
E
C
N
CGA
626
GTG GCC GAG
686
G
Q
D
R
A
E
CTG TGG
L
D
G
A
I
N
L
AGT
S
L
Y
A
V
L
L
P
G
ATC AAC
I
N
GCA TTC
A
746
GGC AAG
G
L
R
Q
A
GAC MG
D
K
N
S
A
W
H
H
I
C
I
G
E
K
L
G
T
T
W
V
F
L
I
AAT
N
I
L
L
G
Q
GTT GGA GAG CTT AGC
V
N
G
ATC
I
E
L
CCC MC
GGG GAG AAC
G
E
D
D
E
Q
CAG TTC
Q
F
N
T
GAC ACT
D
T
I
1166
A
GTG
1226
V
AAC ATA TGG
N
1106
R
CTG GCA
L
1046
Q
1286
W
TGC TCC ACG AAC
ATG CCT
1346
CGA GGG
AAG TGG
1406
A@CSTNMP
GT'C GAT GTG TTT
V
S
T
986
I
GTC GCA CAG
V
926
S
GAG ATT
E
866
K
CTG CGG TCC AGC
W
806
F
TGG CAC CAT ATC TGC ATC ACC TGG ACC ACT CGA
GAG ATC ATC MC
E
V
CTA TAC
Y
V
F
G
G
GCT TCC
A
S
K
W
1472
CTACCTTCTCCCTGTCCCAGAGGCCAA
L
GAGCGGGCTmCTGGGCAGTTCAAGGCATCTATTCCCGAGTTCAACTAAAATffCTGGCCTtACTAGGAAACAACCAG
AGCCCtTMGGCAGGCTGTGTGGCCTCCrrTGTCTTAGGCTCCTATG~CTTAffGC~TGTTCTTTGGTGGGAAGTtA
CCGAAGCCCTGGGAAGAGTCCTCAGCCAClTCCTGCTGGG~~~A~~~~GTGAGC~~C~CCC~C~~
MATGffAGTGCMCCCAGCCCTGCCTGTCATmGGATCCTTAGTCTCTCGTGTGTGCrrCCCCTC
TGGCTGTGTGGTCATCCTACCGGGGTGGCCTGGGTCCCTTGTG
CACAGMGCTACCCGCCCCTGAMCAGGGTCTCTCCCTCA~~~T~G~~~G~~CTCC~~~GGG~CTG
CAGCTGCTGGAGGGCCACGTGCCCAGACACTCCCCAGCATCCCCCAC
AG~CCCCACCCTCTGTCCACCTCTCAGACCCCACGCTTCTAAGGACCATTGCTGGGFTGGCTTTCMAAGCTGCTGC
TCTCATCTGGTGCCAAAAGTTCATTTGC4GCTTCTACACCGTTCTGGGTrrGGGGATTGACTTTATTCCCCCACAA
AAGAGGAACAGCCATTAGAAGCCAtCCTCCCCTCCTTTTGGC
C9CATTGmTCTGTGAGCATCTGACTCTCCCCCGTCCAGFA
GCTACCTGCTTAGTGACTCCAGGCTGCATCATGTATCATA
ITTTTAMGACAAAGTGATTCAGTGGGGAA
~TAAAGCTATMATATTATATATTTTATTTTTCATACATGTTTAAAGTGCGGATCCATGCATGTTCCATTTGTAGG
ACCAGCTTCACGTGCCCATCCTGACATTGTATGCCACMCAGCTCTTGTGATGGMTTTTGATTMA~GCA~GG
MGATGA,
Figure
E
GAC TTC
GCT GTG CCT GGG CAA GCC AAT
GAG CTG CTT
E
T
N
CCA GGG GCT GTG CTC ATC CTG GGG CAG GAG GIG
P
L
GCT CAC CGG CAG AAG ACA GAG
GAG CTG GAG.AGA
GAA GAG CGT CT-C CTG GAC TTG TAG
E
CTA CGC MG
L
CAG GAC GGG GAG AAG CTG GGC ACC
CCA TGG GTG GAC MC
P
R
GCC CCC CTG CCG AGC
TCC CTC CCT CTC CGT ACA AAC TAC
GAT GGC MG
D
D
HBETSAHRQKTE
GAT GCC ACA CAG CCC TTC
D
E
CTG CAG AGG GTG ACT
L
K
TBASNAGLPSDFR
GGC ACG CCA TTC TCC TAC
CAT CCC ATC MG
GGA AAC ATC ATC
385
S
CCC GAG CTG TAT
GTC AGC
V
L
GAC CGT GTC CTC CCC GCA CM
365
L
CT'C CAC ACC MC
GCA Cl-G
GAG TGG
GGA GGC AGA TTT
345
K
CCA GGC ATC
GAC GGC ATG TGG
305
R
ACG TTG
CTG CCC CTG TTT
285
Q
E
GAC GAG AAG TCC CTG CTC CAC AAT
GFG CTG ATA
265
V
CTC CAG CGG AGG CTG GGG GAG CTG GAG AGG CAG TTG
ATC AAG MG
225
V
CTC GAG CTC CM
GAG GTG
145
H
@
1551
1630
1709
1788
1867
1946
2025
2104
2183
2262
2341
2417
2494
2555
2561
sequence
of Nurp
cDNA
and its predicted
amino
acid sequence.
The last nucleotide
of each line is numbered
to the right. The
I. Nucleotide
translated
protein
sequence
is shown below corresponding
nucleotide
sequence
and is numbered
at the left. The putative
signal
peptide
of 16 amino
acids
is underlined.
A dot indicates
the predicted
first amino
acid of the mature
protein.
Putative
glycosylation
sites are circled. Two putative
ATITA
mRNA
instability
motifs
are present
in the 3’untranslated
region
and are boxed. The putative
polyadenylation
signal
(ATTAAA)
is underlined.
2466
Rat
J. Neurosci.,
April
15, 1996,
76(8):2463-2478
Tsui et al.
l
Narp Expression
and
Regulation
in Brain
Narp
234
81
81
Rat
Narp
284
*
G
-
J-
-
K
Rat
SAP
Rat
CPZ?
Rat
Narp
Rat
SAP
Rat
CRP
180
Rat
Narp
384
Rat
SAP
208
Rat
CRP
211
Rat
Narp
416
Figure 2. Comparison of rat Nurp amino acid sequence with rat CRP, rat SAP, guinea pig Apexin, and human NPII. A, The full-length amino acid
sequences of rat CRP (Rassouli et al., 1992) and SAP (Dowton and McGrew, 1990) are shown with the corresponding homologous region of Nap.
Identical amino acid residues are boxed. The eight amino acid “pentraxin family signature” is marked in bold. The p-strand regions defined by x-ray
crystallography of human SAP (Emsley et al., 1994) are indicated by letters&0 and a single overline above the corresponding residues. Residues involved
in calcium binding are indicated by a dot: Asp-58, Asn-59, Glu-136, Gln-137, Asp-138, and Gln-148. Conserved cysteine residues 36 and 95 are highlighted
by an asterisk. B, Full-length Narp is compared to guinea pig Apexin and human NPII. Regions that are identical in all three sequences are boxed, and
regions identical between two are shaded. Figure 2mcontinues.
pig homolog of Narp (Fig. 2B). The nucleotide homology between
Narp and Apexin continues beyond the end of the ORF into the
3’-untranslated
sequence (82% identity in ORF, 25% identity in
the 5’-untranslated
sequence, and 58% identity in the 3’untranslated sequence), further suggesting that Nalp is the rat
homolog of Apexin. Apexin is expressed at high levels in the
mature
sperm
acrosome and is hypothesized to play a role in
protein
aggregation
during
acrosome
biogenesis.
Regulation
relative
studies
to
transcript
tween
species
termed
clone
testis
size.
Narp
have
but
not
does
Accordingly,
been
not
reported
yield
the
a conclusive
difference
in
this
system.
comparison
transcript
of
size
be-
could be attributable
to differences in
The fourth
novel
pentraxin,
NPII (Fig. 2B; also termed NPTX2), is a human genomic
that was identified
by a low-stringency
screen
with
NP (Hsu
or
and
Apexin
in
tissue-specific
expression.
identical
Perin,
to
and
It is
notable
that
the reported
Apexin cDNA is only 1572 nucleotides
compared
to the Narp cDNA, which
is 2561. The Apexin sequence
appears
to be full-length
because
Northern
analysis
of Apexin
identifies
a prominent
band
of 1.6 kb in testis
(brain
RNA was not
examined)
(Reid
and Blobel,
1994), which
is consistent
with
the
size
of their
reported
cDNA. Our Northern
blot
(see
below)
indicates that the Nurp mRNA transcript is enriched in brain
functional
1995). NPII-predicted
amino acid sequence is 94%
Narp. This degree
of identity,
together
with
its similar
mRNA size and distribution of expression, suggests that NPII may
be the
human
homolog
of Narp. Consistent
with
its possible
regulation
as an IEG, we note
that
the putative
promoter
region
of NPII possesses consensus binding
sites for the transcription
factors CREB and Zif268.
and
Nat-p mRNA is enriched in brain and is rapidly induced
by seizure
The expression of Narp mRNA was examined by Northern blot
analysis of total RNA from rat cerebral cortex, hippocampus, and
different peripheral tissues (Fig. 3). We also assayed Narp mRNA
in hippocampus
and
cortex
after
MECS. Narp is enriched
in
cortex
and hippocampus
relative to peripheral
tissues. Lower
levels of Narp mRNA were also detected in the testis (and ovary;
data
not
shown).
Narp mRNA is induced in the hippocampus
within 1 hr after MECS and remains elevated for as long as 8 hr.
Densitometry of the autoradiogram
indicates that Narp mRNA is
induced at least fivefold by MECS. Increased levels of mRNA in
the hippocampus
after
MECS are associated
primarily
with
granule
cell
neurons
(Fig. 4). Nurp mRNA is induced in animals
Tsut et al.
l
Narp
Expression
and
Regulation
in Bram
J. Neuroscl.,
April
15, 1996,
76(8):2463-2478
2469
Narp
Apexin
NPTXZ
Nary,
Apexin
NPTXZ
100
99
98
Narp
Apexin
NPTX2
Narp
LGEL ERQLLRKVAE
Apexin
NPTX2
Narp
Apexin
NPTX2
LNALLQRV
LNALLQRV
TELERGNSAF
TELERGNSAF
KSPDAFKVSL
KSPDAFKVSL
LEDEKSLLHN
TNYLYGK
TNYLYGK
ETSA
IKKTLPEL
IKKTLPEL
200
193
198
250
243
248
Narp
Apexin
NPTX2
300
293
298
Narp
Apexin
NPTX2
350
343
348
Narp
Apexin
NPTX2
LILGQEQDTV
GGRFDATQAF VGELSQFNIW
Narp
Apexin
NPTX2
DRVL
400
393
398
432
425
430
Figure 2 Continued
pretreated with cycloheximide,indicating that the induction does
not require new protein synthesis(data not shown).mRNA induction in the absenceof new protein synthesisis a defining
characteristicof IEGs (Lau and Nathans,1987).
Narp is rapidly regulated by physiological
synaptic activity
The regulation of Nulp mRNA was also analyzed usingthe paradigmof in vivo LTP. As shownin Figure 4 (fop), Nalp mRNA is
strongly induced4 hr after an HF synapticstimulusthat produces
LTP (n = 2). The time courseof Nurp mRNA increasesafter LTP
induction was also assayed.Increasesin Narp mRNA were detected asearly as30 min (n = 6), appearednear-maximalafter 1
hr (n = 2), 2 hr (n = 2) and 4 hr (n = 2), and returned to basal
level after 24 hr (n = 2) (data not shown). This time course is
similar to that after MECS (Fig. 3). Induction of Narp mRNA
after LTP is dependent on the activation of NMDA receptors
becauseprevious treatment of rats with the noncompetitive
NMDA receptor antagonistMK-801 (1 mg/kg, i.p.) blocked increasesin both Nuvp mRNA and LTP (n = 3; data not shown).
Nurp mRNA is readily detected by in situ hybridization in
normal cortex and is enriched in cortical layers 2/3 and 5/6. To
determinewhether this basalexpressionis regulated by synaptic
activity, we monitored Nurp mRNA levels in visual cortex after
monocularinjection of TTX. This manipulationreducesafferent
activity to the contralateral visual cortex and resultsin a rapid
decreasein the expressionof several of the IEGs known to be
expressedin cortex, indicating that their basalexpressionis continuously maintainedby natural afferent synapticactivity (Worley
et al., 1991). Rapid responsegenesthe expressionof which is
dynamicallyregulated in this paradigminclude~$268 (Worley et
al., 1990,1991),prostaglandinsynthase(Yamagata et al., 1993),
Egr-3 (Yamagataet al., 1994a),Arc (Lyford et al., 1995),and PA
activin (Andreassonand Worley, 1995). Becausethere are no
intracranial manipulationsandbecausethe perturbation resultsin
2470
J. Neurosci.,
April
15, 1996,
76(8):2463-2478
Tsui et al.
3. Narp mRNA induction in hippocampus and cortex by MECS
and expression in peripheral tissues. Northern analysis of Narp mRNA
after an MECS-induced seizure in the hippocampus and cortex and
expression in peripheral tissues. In the hippocampus, NaT mRNA is
induced as early as 1 hr after MECS, and expression remains elevated for
8 hr. Narp mRNA is also induced by MECS in the cortex. Narp mRNA is
enriched in control hippocampus and cortex (highlighted with a dot)
relative to peripheral tissues.
Figzwe
a reduction of natural activity rather than a stimulation of activity,
as results with MECS and in viva LTP, dynamicregulation in this
monoculardeprivation paradigmprovides someof the strongest
evidence of a role for the gene in normal synaptic physiology. As
demonstratedby Figure 5, Nalp mRNA is reduced in the deafferented visual cortex, most noticeably in cortical layers 213 (top),
within 4 hr after TTX injection. The reduction of Nalp mRNA is
mostevident in comparisonsof the contiguousassociationcortex
and primary visual cortex where there is a prominent
reduction
of
hybridization. In control cortex, the level of hybridization is uniform acrossthis sameboundaryregion. Becausevisualprojections
in the rodent are -90% crossed(Zilles et al., 1984) the effect of
deprivation
is almost exclusively
contralateral.
The
effect of monocular‘ITX injection on basalexpressionof Narp
mRNA is lessdramatic than is evident with certain other IEGs
suchaszif268. This differencein the degreeof reduction of Narp
andzif268 mRNAs doesnot appearto be attributable to a slower
rate of degradationof Narp becausesimilar differences in the
degreeof mRNA reduction were alsoobserved18 hr after ITX
injection (n = 5; data not shown).Narp mRNA in cortex is less
abundant
than zif268, and technical differences
in detection
may
contribute to their differential responsiveness.
Alternatively, basal
expressionof Narp in cortex may be regulated by signalsin
addition
to those generated
by afferent activity.
mRNA expression is developmentally regulated
and enriched in limbic structures and sensory ganglia
The developmentalexpressionof Narp mRNA was assayedby
Northern blot analysisof total RNA isolatedfrom whole brainsof
embryonic day 14 to adult (Fig. 6A). Narp mRNA was first
detected at embryonic day 14 and increasedmonotonically to
peak levels at postnatal day 21. Levels remain high in adult
animals.In situ hybridization wasusedto examinethe anatomical
distribution of Narp mRNA in embryonic day 19 rats. Nalp
mRNA is detected in the habenula,dorsalmedial hypothalamus,
hippocampus,and trigeminal ganglia (Fig. 6B). Lower levelsare
Narp
Narp Expression
and
Regulation
In Brain
cortex
Hippocampus
monocular
l
4. Narp mRNA in hippocampal granule cells by LTP stimulation.
Rats were chronically implanted for in viva recording and were stimulated
as described in Materials and Methods. Natp mRNA was analyzed by in
situ hybridization. The top brain is from a rat that received a unilateral
high-frequency LTP stimulus to the right perforant path and an identical
number of low-frequency stimuli to the left hippocampus 4 hr before
killing. Narp mRNA is markedly increased in the granule cell layer that
received the LTP stimulus. The bottom brain is a composite of half brains
from a naive control rat (left) and a rat that received a MECS seizure 4 hr
before killing (right). MECS results in Narp mRNA induction in the
superficial and deep layers of the neo- and pyriform cortices as well as the
dentate gyrus. Densitometry of the autoradiographic image indicates a
2.5-fold increase in the grain density over the granule cell layer of the
dentate gyrus in the 4 hr LTP hippocampus compared with control.
Figure
presentin cortex and other forebrain structures.A more detailed
analysis of the developmental expressionwill be published
separately.
In vitro synthesis and post-translational modification
of Narp
Zn vitro TNT wasusedto confirm the predicted ORF. The longest
cDNA clone produceda protein with an M, of -46 kDa, which is
identical to the predicted size(Fig. 7A). TNT usinga cDNA clone
that begins-30 nucleotides3’ to the predicted initiator methionine produced an -43 kDa protein @‘-truncated Narp), which
correspondsto the predicted product size synthesizedfrom the
first internal methionine(Fig. 7A). Restriction of the samecDNA
before TNT with BglI, which cuts 67 nucleotides 5’ to the C
terminus of the predicted ORF (nucleotide 1242), results in a
product of reducedsizeconsistentwith the position of the restriction sitein the ORF (data not shown).Theseexperimentssupport
the predicted ORF.
The ORF of Nalp encodesa putative secretory signalpeptide.
To assess
the presenceof a functional signalsequence,full-length
Narp wasprepared by TNT in the presenceof dog microsomes.
The microsomepreparation hasthe necessaryactivity to translocate the nascentprotein into the microsomeand cleavesthe signal
sequence.Narp prepared in the presenceof dog microsomes
yields severalproductsthat migrate on SDS-PAGE with an M, of
-44 to -58 kDa. The -44 kDa product is slightly smallerthan the
-46 kDa product prepared without microsomes (Fig. 7B), consistent with microsome-dependent
signal peptide cleavage. Demonstration that Nalp is secretedby COS-1 cells (below) is also
consistentwith the hypothesisthat Narp possesses
a functional
signalsequence.
Tsui et al.
l
Narp
Expression
and
Regulation
in Brain
J. Neurosci.,
A
Pmnatal
Tissues
April
15, 1996,
Postnatal
76(8):2463-2478
2471
Tissues
18s
Figure 5. Regulation of Nap mRNA by natural synaptic activity in visual
cortex. Postnatal day 21 rat received a monocular injection of TTX 4 hr
before killing. In situ analysis of Nurp mRNA demonstrates reduced
expression in the deafferented visual cortex (between arrowheads, top). A
prominent difference in expression is evident between contiguous primary
and association visual cortices (boundary is indicated by the lateral arrow),
whereas in control cortex Nurp mRNA expression is uniform across this
boundary. Densitometry of the autoradiographic image indicates a 1%
fold higher grain density over the temporal cortex relative to the contiguous deprived visual cortex. Identical results were observed in four additional 21-d-old rats and three adult rats killed 4 hr after TTX injection.
Narp has three potential N-glycosylation
sites. Other pentraxins
are known to be glycosylated and this modification may be important in their saccharide-binding
properties
(Emsley et al.,
1994). In addition to the product that migrates slightly faster than
Narp prepared without microsomes described above (-44 kDa), a
second product is observed that migrates with an M, of -58 kDa.
To determine whether mature Narp is glycosylated,we examined
the effect of treating the TNT/microsomeproduct with endoglycosidaseH. As shownin Figure X, endoglycosidase
H converts
the -58 kDa band to an -44 kDa product, consistentwith the
interpretation that the -58 kDa band is glycosylatedNap.
Narp protein is secreted
or
COS-1 cells were transiently transfected with pRK.5 Nap
vector alone. Forty hours after transfection, cellswere metabolically labeledwith [““Slmethionine 3 hr before samplingmedia.
Conditionedmediafrom cellswere analyzed by SDS-PAGE and
autoradiography.A prominent band of -58 kDa is presentin the
mediaof cellstransfectedwith pRK5 + Nalp that is not present
in cellstransfectedwith vector alone(Fig. &i). The -58 kDa band
is enriched in the media and was not detected in the pelleted
COS-1cells(data not shown).To confirm that the -58 kDa band
representsNarp, we performed immunoprecipitationsfrom the
conditioned media using a polyclonal rabbit antiseragenerated
againsta full-length Nalp bacterial fusion protein and demonstratedthat the -58 kDa band is selectivelyprecipitated (Fig. 8s).
Thesedata strongly suggestthat Narp is secretedinto the media
6. Developmental regulation of Nqv mRNA expression. A,
Northern analysis of total RNA (10 pg/lane) prepared from forebrains of
embryonic (E) days 14, 15, 17, 19, and 21, and postnatal (P) days 1, 4, 8,
12,16,5 week-, 8 week-, and 3-month-old rats. Nurp mRNA is detected as
early as El4 and increases steadily to peak levels between P16 and P21.
The slight difference in motility of the E21 RNA sample may be attributable to excess salt in this preparation. B, In situ analysis of expression of
Nurp mRNA in El9 rat forebrain (magnification, .5X). Nurp mRNA is
detected in the hippocampus (h), habenula (b), ventromedial hypothalamus (v), and the trigeminal ganglia (t). C, Cresyl violet counterstaining of
the in situ section of El9 rat.
Figure
by the transfected COS-1 cells. Although it remains formally
possiblethat Narp is passivelyreleasedinto the mediaby COS-1
cells that are injured or dying, this is unlikely becausethe metabolic label, which is incorporated into newly synthesizedprotein,
is added40 hr after transfectionat a time when cytotoxic effectsof
this manipulation
should be resolved. Additionally,
media is sam-
pled 3 hr after addition of the metaboliclabel, thereby limiting the
interval during which cellsmight undergospontaneouscell death
and releaseof intracellular contents.
Biochemical studies of Narp: demonstration of
calcium-dependent binding to a complex
saccharide matrix
Pentraxins bind specifically to derivatized saccharides,and this
lectin property is thought to be essentialfor their biological
function (Emsley et al., 1994). The close homology of Narp to
other pentraxins, particularly at the Ca’+- and carbohydratebinding domains, suggeststhat Narp may also function as an
endogenouslectin. Substratestypically usedin pentraxin-binding
assaysare modified agar. Agar is a polysaccharidethat contains agarobiose,sulfate, and pyruvate (Hind et al., 1984), and
agaroseis prepared from agar by removing sulfated sugars.The
2472
J. Neuroscl.,
April
15, 1996,
76(8):2463-2478
Tsui et al.
l
Narp Expression
and
Regulation
in Brain
C
kDa
EndoH
-
+
ld)a
-68
-18
l 18
Figure Z In vitro synthesis of Nurp protein: post-translational modification of Nurp protein by dog, microsomes. A, Circular and linearized Nary cDNA
plasmids subcloned in pBluescript SK+ vector were used as templates to synthesis l\iarp pro&in b; tn vifro TNT. Translated ““S-labeled prod&s were
analyzed by SDS-PAGE. An -46 kDa protein product is detected using the circular or linearized full-length Num1 olasmids
farrow‘l.
An -43 kDa
~~~ nrotein
r~ - .-...
I
\
is synthesized from a plasmid, .5’-trundated N&u, that lacks the puta&e initiator methionine but includes the first internal methionine. The observed
product size is consistent with translation initiation from the first internal methionine. Two bands, -34 and -27 kDa, are nonspecific products from the
pBluescript vector alone (data not shown). No product is detected in the absence of added DNA (d-H,0 control). B, Cleavage of the putative signal
sequence of Nurp protein by dog microsomes. Full-length Nurp was synthesized by in vitro TNT reactions in the presence or absence of dog microsomes
(DM). Translation is more efficient in the presence of microsomes, and nonspecific bands at -34 and -27 kDa are not evident. A slight reduction in the
size of the Nulp protein is detected compared to the Nurp protein synthesized in the absence of dog microsomes, consistent with cleavage of the signal
sequence (arrowheads).
C, N-glycosylation of Nurp protein. Nurp prepared by in vitro synthesis in the presence of dog microsomes yields major products
of -58 and -44 kDa (arrows). Incubation of Nurp with 2 mU of endoglycosidase H shifts the -58 kDa protein to -44 kDa, supporting the inference
that the -58 kDa band is a glycosylated form of Narp.
polysaccharide
backbone of agarose (marketed
under trade
name Sepharose) can be derivatized by covalent attachment to
a variety of molecules. CRP and SAP are known to bind
to phosphatidylethanolamine-Sepharose
(PE-Sepharose)
and
phosphatidylcholine-Sepharose
(PC-sepharose),
and these substrates are commonly used to purify CRP and SAP from body
fluids (Schwalbe et al., 1992). In all cases, binding to these substrates is calcium-dependent.
To examine the hypothesis that
Nurp is a calcium-dependent
lectin, a full-length C-terminal myctagged Nalp expression construct was prepared in pRK5 and
expressed in COS-1 cells for binding studies. The COS-1 cellderived myc-tagged Narp is biologically
active in the neurite
outgrowth assay described below and was used in preference to
native Narp because of the relative insensitivity of the Narp
antisera for immunoblots.
As demonstrated
in Figure 9, myctagged Narp binds specifically to agar in the presence of Cazt and
quantitatively elutes in the presence of the divalent cationic chelator EDTA. The estimated size of the EDTA-elutable
band is
-60 kDa and corresponds closely to the size of the glycosylated
Nalp prepared by TNT with dog microsomes. No EDTA-elutable
band was detected in parallel experiments that used conditioned
media from COS-1 cells transfected with the expression vector
lacking the Narp insert. Myc-tagged Nalp did not bind in a
calcium-dependent
manner to agarose,
Sepharose, or PC-Sepharose (data not
substrate specificity of Narp is distinct
The difference in binding to agar and
pyruvate or sulfated side chains present
in binding to Narp.
low-melting agarose, PEshown), indicating that the
from either CRP or SAP.
agarose suggests that the
in agar may be important
Narp promotes neurite outgrowth of cortical
explant neurons
Pentraxins possess structural and biochemical similarities to the
class of calcium-dependent
plant-derived lectins that include concanavalin A (Emsley et al., 1994). Concanavalin
A produces
marked effects on a variety of cell types including neurons (Lin
and Levitan, 1987, 1991), and we have examined the possibility
that Nalp may mimic certain of these effects. Concanavalin A is
highly active in promoting neurite outgrowth (Lin and Levitan,
1987). Accordingly, we examined the possibility that Nalp might
also promote neurite outgrowth.
Cortical explants were prepared from postnatal day 1 rat pups
as described previously. One day after plating cortical explants,
COS-1 cells, which had been transfected previously with either a
Narp expression construct or the vector alone, were added to the
culture. In cocultures expressing Narp, cells at the periphery of the
TSUI et al.
l
Narp Expresslon
and
Regulation
J. Neurosci.,
m Bram
B
A
xP 2uQ
kDa
kDa
-200
-97
-68
4
-43
-29
-29
-18
-14
Supernatant
-14
lmmunoprecipitation
Figure 8. Transient expression and secretion of Nurp by COS-1 cells.
COS-1 cells were transientlv transfected with 10 ~g of plasmid DNA
(pRK5 vector alone or pRR5 + Nqu). Forty hours after transfection,
COS-1 cells were labeled with 250 uCi/ml 1”‘Slmethionine for 3 hr in
Met- DMEM supplemented with 1’% dialyied’fetal bovine serum. A,
Supernatants from pRK5 vector alone and pRJS5 + Nq were collected
and fractionated on SDS-polyacrylamide gels. The armwhead indicates an
-58 kDa band expressed only by COS-1 cells transfected with pRK5 +
Narp, suggesting that Nurp is secreted into the media. B, lmmunoprecipitations’were performed using polyclonal antisera generated against the
recombinant bacterial protein as described in Materials and Methods. The
-58 kDa band is selectively immunoprecipitated by immune serum.
explant exhibited exuberant outgrowth of processes
within 24 hr
of the addition of the COS-1 cells. Processoutgrowth was observedsurroundingthe perimeter of each of 26 separateexplants
from three dishesin cocultures that expressedNurp, whereas
explantscultured with control COS-1 cells (17 separateexplants
from 3 dishes)or without COS-1 cells(16 explantsfrom 3 dishes)
April
15, 1996,
76(8):2463-2478
2473
exhibited no processes.By 48 hr in coculturewith Narp, processes
were longer and more prominent than at 24 hr, and isolatedcells
with elongatedprocesses
were seensurroundingthe explant that
appearedto have migrated out of the body of the explant. In
control cocultures,only infrequent short processeswere present
by 48 hr without evidence of cellular migration. Similar results
were obtained in four additional experiments.As a control for the
specificity of Nalp in this assay,we examinedthe effect of coculturing cortical explantswith COS-1 cellsthat expressand secrete
amyloid precursor protein-like protein (APLP-2) (Slunt et al.,
1994).Theseexplantsappearedidentical to control explantscultured either without COS-1 cellsor with COS-1 cellstransfected
with vector alone (data not shown).
To determine the cellular compositionof the Narp-dependent
processoutgrowth, we performed immunohistochemistryusing
antiserafor the neuron-specificproteins MAP2 (Pennypackeret
al., 1991), which is selectively expressedin neuronal dendrites,
and tau, which is expressedin neuronal axons (Binder et al.,
1985),aswell asfor the glial-specificprotein GFAP (Rinamanet
al., 1993). In control explants, MAP2-positive cell bodies and
processes
werepresentwithin the explant, and few processes
were
seen extending beyond the edge of the explant (Fig. l&4). By
contrast, explants cocultured with Nary secreting COS-1 cells
exhibited MAP2-positive cells with exuberant processesboth at
the border of the explant and extendingmanycell body diameters
from the edge of the explant (Fig. 1OB). The morphology and
immunohistochemicalproperties of thesecellsindicate that they
are neurons.Tau immunostainingwasdifficult to detect in control
explants and was not increasedin explantscocultured with Narp
secretingCOS-1 cells (data not shown).GFAP-stained glial cells
appearedto be largely restricted to the body of the explant at the
24 and 48 hr time points (data not shown). We concludefrom
theseexperimentsthat Nalp-secretingCOS-1 cellspromote neurite outgrowth andmigration of neuronsfrom the cortical explant.
This effect is not accompaniedby prominent growth or migration
of glial cells, suggestingthat the effect is primarily on neurons.
We usedthe neurite ,outgrowth promoting effect of Nalp as a
bioassayof its function to test the activity of C-terminal myctagged Narp. Cortical microexplants grown in coculture with
COS-1 cellsexpressingmyc-Nalp demonstratedthe samegrowth
of neurites and migration of neuronsas explantscoculturedwith
COS-1 cells secretingnatural Nurp (data not shown). We conclude that the myc epitope tag doesnot interfere with the biological activity of Narp in this assay.
Using the COS-1 cell-generatedmyc-Nurp, we performed two
additional setsof experimentsto confirm that the active principle
in the coculture paradigmis Narp. In the first set of experiments,
agar column
chromatography
was used to prepare
partially
puri-
fied myc-taggedNurp. Addition of this material to the microexplant culture reproducedthe neurite outgrowth effect of coculture
with Narp-secretingCOS-1 cells (Fig. lL4). In control experiments,identical agarcolumn fractionspreparedfrom COS-1cells
transfectedwith the pRK5 vector alone did not induce neurite
outgrowth.
The secondsetof experimentsuseda myc monoclonalantisera
to immunodepletemyc-taggedNurp from the agar columneluate.
Addition of myc mAb and subsequent precipitation with
G-protein-Sepharoseremoved neurite outgrowth promoting activity (Fig. 11B). In control experiments,addition of mouseascites
fluid followed by G-protein precipitation did not block this
activity.
In the final set of experiments,we sought to establishthe
2474
J. Neurosci.,
April
15, 1996,
16(8):2463-2478
Tsui et al.
l
Narp
Expression
and
Regulation
in Brain
kDa
kDa
43.6 -
-
70.8
-
43.6
-
28
Figure 9. Ca’+-dependent binding of Narp protein to an agar column. Narp was synthesized by COS-1 cells transiently transfected with an expression
vector for myc-tagged Narp. A shows the Western blot analysis using mouse anti-myc Abs (1:500) of the COS-1 cell supernatant @e-flow Through),
flow-through fraction, wash fractions (0.15 M NaCI, 0.05 M Tris, pH 7.4), EDTA elution fractions (0.15 M NaCl, 0.05 M Tris, 0.02 M EDTA, pH 7.4), and
SDS-loading buffer fraction. Myc-tagged Narp protein (arrowhead; -60 kDa) binds to the agar column in a Ca*+ -dependent manner and is quantitatively
eluted in the second and third EDTA elution fractions. Serum contains proteins that cross-react during development of the anti-myc immunoblot and are
seen in the Pre-flow Through and Flow Through fractions. B, Identical agar column fractions from conditioned media of COS-1 cells transiently transfected
with the pRK5 vector alone. Note similar cross-reacting bands in the Pre-flow Through and Flow Through lanes but the absence of EDTA-elutable
myc-immunoreactive protein.
concentration
of Narp necessary to promote neurite outgrowth.
Again, we used myc-tagged Narp and established a direct competitive ELISA. Aliquots of agar column fractions were assayed in
parallel for biological activity, and levels of myc-tagged Narp were
quantitated. These studies indicate that full activity is produced by
-40 @ml myc-tagged Narp (Fig. 11B). Based on our estimated
molecular weight of the Narp monomer (-58 kDa), this translates
to an effective concentration
of -0.8 nM. Higher concentrations
were without additional effect.
In conjunction, these experiments establish that Narp promotes
neurite outgrowth and establishes the concentration
range of its
activity to be comparable to that of growth factor-induced
effects
in other systems.
Chromosomal localization of Nay
The mouse chromosomal
location of Narp was determined
by
interspecific backcross analysis using progeny derived from matings of [(C57BL/6J X Mus spretc~)F, X C57BL/6J] mice. The
mapping results indicate that Narp is located in the distal region of
mouse chromosome 5 linked to Epo, Pd&a, and Flt3. Although
159 mice were typed for every marker, up to 191 mice were typed
for some pairs of markers. Each locus was analyzed in pairwise
combinations for recombination
frequencies using the additional
data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each
pair of loci, and the most likely gene order is: centromere - Epo 71191 - Pdgfa - 31177 - Flt3 - O/l60 - Narp. The recombination
frequencies [expressed as genetic distances in centiMorgans
(CM)
+ SE] are: Epo, 3.7 k 1.4; Pdgfa, 1.7 + 1.0 [Flt3, Nalp]. No
recombinations
were detected between Flt3 and Narp in 160
animals typed in common, suggesting that the two loci are within
1.9 CM of each other (95% confidence limit).
We have compared our interspecific map of chromosome 5 with
a composite mouse linkage map that reports the map location of
many uncloned mouse mutations [compiled by M. Davisson, T.
Roderick, A. Hillyard, and D. Doolittle and provided by GBASE,
a computerized
database maintained at The Jackson Laboratory
(Bar Harbor, ME)]. Nalp mapped in a region of the composite
map that lacks mouse mutations with a phenotype that might be
expected for an alteration in this locus.
The distal region of mouse chromosome 5 shares a region of
homology with human chromosomes 7 and 13.
DISCUSSION
We have identified and characterized a novel IEG, termed Narp,
that is a member of the pentraxin family. Nurp mRNA is rapidly
and transiently induced in neurons by electrically evoked seizures.
Narp is also dynamically regulated by physiological synaptic activity in the adult hippocampus and visual cortex. In the hippocampus, synaptic induction of Nav is associated with the production
of LTP and is dependent on NMDA receptor activation. In this
paradigm, Nulp mRNA induction, like the induction of other
IEGs, is not simply linked to synaptic activity but, rather, to
specific forms of activity that activate the NMDA receptor (Worley et al., 1993). Identical numbers of synaptic stimuli adminis-
Tsui et al.
l
Narp
Expression
and
Regulation
m Brain
J. Neurosci.,
April
15, 1996,
76(8):2463-2478
2475
initial reports that is suggestiveof function. Biochemicalcharacterization of Nalp protein demonstratesthat it binds to agar, a
complex polysaccharidematrix, in a calcium-dependentmanner.
Calcium-dependentbinding to sugarsis a shared property of
biochemicallycharacterizedmembersof the pentraxin family and
is consistentwith the high degreeof conservationbetweenNalp
and other pentraxinsin the essentialdomainsinvolved in calcium
and saccharidebinding. The binding specificity of Narp appearsto
be distinct from that of CRP or SAP in that it bindsagarbut not
phosphatidylcholine or phosphatidylethanolamine derivatized
agarose.The physiologicalsaccharidespecificityof Narp in brain
remainsto be determined.Like other pentraxin family members,
Narp possesses
a functional signalsequenceand is secretedinto
the media when expressedby COS-1 cells. Presumably,Natp is
also secretedby neurons,consistentwith a possiblefunction in
intercellular signaling.
An interesting aspectof the pentraxin family is that it shares
secondaryand tertiary structurewith the plant lectin concanavalin
A (Emsleyet al., 1994).Although the precisecellular functionsof
membersof the pentraxin family remain controversial,their multivalency and calcium-dependentlectin properties appear to be
important. CRP is thought to play a role in non-Ab-mediated
immune responsesby binding and aggregatingbacteria or other
pathogens(Siegel et al., 1974, 1975). SAP is believed to coat
moleculespresent in the basementmembraneand protect them
from proteolytic degradation (Emsley et al., 1994). SAP is also
reported to be a component in all known forms of amyloid,
including the amyloid of Alzheimer’s disease,and may therefore
play a role in certain diseaseprocesses(Coria et al., 1988;Pepys
et al., 1994). Evidence that the lectin property of pentraxins is
central to their function supportsthe hypothesisthat Narp may
alsofunction in vivo asan endogenous,calcium-dependentlectin.
Severallines of evidencesuggesta role for specificinteractions
betweenlectins and glycosylatedproteins or lipids in neurobiolFigure IO. Nurpinduces
neuriteoutgrowthfromcorticalexplantcultures. ogy. Complexoligosaccharideantigensand carbohydrate-binding
Corticalexplantspreparedfrompostnatalday1 rat pupsw&e cocultured
proteins have been localized to specific developing neurons
for 48hr withCOS-1cellsthat hadoreviouslv
withaNuro
* beentransfected
1
(Dodd et al., 1984;Dodd and Jessell,1985;Blum and Barnstable,
expression
construct(B) or thevector alone(control;A) asdescribed
in
MaterialsandMethods.Cultureswerestainedwith the neuron-specific 1987). Endogenous“soluble lectins” have been purified from
anti-MAP2mAb. In explantscoculturedwith controlCOS-1cells(A),
brain and peripheraltissues(Kobiler et al., 1978;Barondes,1984)
MAP2-positivecellsare largelyrestrictedto the bodyof the explant.By
and are capableof aggregatingcells, and they can alter growth
contrast,exuberantMAPZpositiveneuriteoutgrowthisevidentfrom the
properties
of neuronsin culture (Joubert et al., 1987;Kuchler et
corticalexplantscocultured
with COS-1cellsexpressing
Narp (B). MAPZ
and coworkers have characterized a soluble
positivecellbodiesarevisualized
beyondthe edgeof theexplant,suggest- al., 1988). Jesse11
ingmigrationfrom the explant.Magnification,400X.
lectin, termed RL-14.5, that is expressedby neuronsin the developing spinal cord, brainstem,and dorsal root ganglia(Regan et
al., 1986; Hynes et al., 1990). These studiessupport a role for
tered at low frequency, which do not activate the NMDA receplectins in the developmentof specificcellular interactionsin the
tor, do not induce Narp. Also like other IEGs, Narp mRNA is
expressedat relatively high levels by neuronsin the adolescent nervous system.
Solublelectins,purified from either brain or peripheraltissues,
and adult neocortexwhere natural synaptic activity is dependent,
are highly homologous,and their saccharidebinding is indepento a substantialdegree,on NMDA receptor activation (Miller et
dent of calcium(Barondes,1988;Clerch et al., 1988;Hynes et al.,
al., 1989;Fox and Daw, 1993).Basalexpressionof Narp in cortex
1990). Narp is structurally distinct from the family of soluble
is dependenton natural synapticactivity as demonstratedby the
lectins,and its lectin property iscalcium-dependent.Studiesusing
rapid reduction in expressionin the primary visual cortex after
plant lectins, which as noted are structurally and biochemically
interruption of afferent visual input. Accordingly, Nav is exrelated to pentraxins, support a role for this subclassof calciumpressedby neuronsand is dynamically regulated in responseto
dependentlectinsin neurobiology.Binding sitesfor concanavalin
specifictypes of synaptic activity that are effective in producing
A and pea lectin are enriched in the developingand adult brain
long-term changes in cellular and synaptic properties. Nalp
mRNA is alsoexpressedearly in the developingnervous system and are associatedwith specific neuronal and glial populations
and is enriched in neurons of the limbic system and sensory (Simpsonet al., 1977;DeGrauw and Liwnicz, 1986;Waters et al.,
1990;Adam et al., 1993).Plant lectins modify the rate of desenganglia,suggestinga role in early stagesof neuronal development
sitization of glutamatereceptor channelsin both invertebrate and
that is not presentlyknown to be driven by synapticactivity.
vertebrate systems(Kehoe, 1978; Shinozaki and Ishida, 1979;
Narp appearsto representthe rat homologof humanNPII and
Huettner, 1990; Thio et al., 1993). In the rodent hippocampus,
guineapig Apexin. However, there islittle information from these
2476
J. Neurosci.,
April
15, 1996,
76(8):2463-2478
Tsui et al.
l
Narp
Expression
and
Regulation
in Brain
60
VECTOR
Fluid Ascites
ALONE
0
50 pl
100 pl
200 pl
(20)
(40)
(60)
400 pl
(80)
Anti-myc
Abs
Narp rig/ml
Il. Neurite outgrowth assaywith partially purified myc-tagged Nurp. A, Partially purified myc Nurp induces neurite outgrowth. Cortical explants
from postnatal day 1 rat pups were cultured on poly-L-ornithine-coated plates. Myc-tagged Nalp was partially purified from COS-1 cell supernatant, and
aliquots were added to culture media. The amount of myc-tagged Nurp was determined by ELISA. After 24 hr, the number of explants with neurite
outgrowth was determined and expressed as the percent of the total number of cortical explants. Data are from three separate experiments, each
performed in duplicate. Ncurite outgrowth is observed with -40 ngiml Nurp. Control experiments used identical volumes of agar column fractions
prepared from conditioned media of COS-1 cells transfected with vector alone. Explants treated with the control fractions demonstrate a 20-30%
spontaneous outgrowth. B, Immunodepletion with myc Ab blocks neurite outgrowth activity. Partially purified myc-tagged Nurp (-60 ng in 200 ~1) was
immunodepleted using mouse anti-myc Ab conjugated to G-protein-agarose beads. Control experiments substituted mouse ascites for anti-myc Ab.
Immunodepleted and control fractions were added to media, and neurite outgrowth was assayed after 24 hr. Data are from two separate experiments,
each perfoimed in quadruplicate.
Figure
several plant lectins, including concanavalin A, selectively block
desensitization
of the AMPA receptor without altering NMDA,
GABA,,
or nicotinic acetylcholine receptor responses (Thio et
al., 1992). Evidence that glutamate receptor function can be
selectively modified by plant lectins provides a precedent for
understanding
how Narp might function on a molecular level.
Narp is unique among the known set of endogenous
lectins
present in neural tissues in that it is regulated as an IEG and that
its expression is tightly linked to physiological neuronal activity.
Studies examining the cellular function of Narp demonstrate
that it promotes neuronal dendritic outgrowth and migration of
neurons from cortical explant cultures. These observations suggest that Nalp may function as a novel, neuron growth-promoting
molecule that is tightly coupled to physiological activity. In this
regard, Nay is similar to more conventional growth factors such
as brain-derived
growth factor or nerve growth factor, which are
also rapidly regulated by neuronal activity (Bachoo et al., 1992;
Patterson et al., 1992). Although the mechanism of the effect of
Narp has not been determined, there are several clues as to how
it might act. The effect of Narp is most obvious when microexplants are grown on a poly-t.-ornithine
substrate, which is more
supportive of neurite outgrowth than untreated plastic or glass but
less supportive than poly-L-lysine, collagen, or NCAM. In our
assays, Narp did not appear to promote neurite outgrowth when
explants were cultured on collagen. Accordingly, it is possible that
Narp is simply a better substrate for growth than polyornithine.
Interactions between the extracellular matrix and membrane receptors of the integrin family can alter cell growth properties via
modulation of intracellular
kinase-dependent
mechanisms similar
to conventional receptor tyrosine kinases (Bachoo and Polosa,
1991). The major argument against the notion that Narp acts via
a substrate effect is its potency. The potency of Narp is remarkable
in that 40 r&ml (0.8 nM) is fully effective in promoting neurite
outgrowth, whereas substrates such as NCAM and laminin are
used in the concentration range of 1 mgiml (Lemmon et al., 1992).
Moreover, we have been unsuccessful in reproducing the growthpromoting
effects of Nalp by pretreating
either plastic- or
polyornithine-coated
substrates with the same preparations
of
Nalp that are effective when added to explants in media (our
unpublished
observations). Finally, we note that concanavalin A
and pea lectin have similar, marked substrate-dependent
effects
on neuronal process outgrowth and are effective at concentrations
in the range of 10 nM to 1 PM (Lin and Levitan, 1987; Carrow and
Levitan, 1989; Lin and Levitan, 1991; Outenreath
and Jones,
1992). Based on similar arguments as presented for Narp, it is
believed that the growth-promoting
effects of plant lectins are
attributable to the binding of specific receptors on the surface of
neurons and are not attributable to a substrate effect. Pentraxins
and plant lectins are multivalent lectins, and their binding efficacy,
as well as activity in physiological assays, is dependent on their
multivalency (Lin and Levitan, 1991). Accordingly, Narp might
bind to glycoprotein targets on the cell surface to effect clustering
of receptors or changes in cell-cell or cell-substrate interactions.
In either event, whether it is mediated by a substrate effect or by
a more classical receptor-mediated
mechanism, the growthpromoting effects of Narp, in combination
with its dynamic expression in the brain, suggest an important role in developmental
and activity-dependent
plasticity.
REFERENCES
Adam E, Dziegielewska KM, Saunders NR, Schumacher U (1993)
Neuraminic acid specific lectins as markers of early cortical plate
neurons. Int J Dev Neurosci 11:451-460.
Agranoff B (1981) Learning and memory: biochemical approaches. In:
Basic neurochemistry, 3rd Ed, p 801. Boston: Little & Brown.
Tsui et al.
l
Narp Expression
and
Regulation
in Brain
Andreasson
K, Worley P (1995) Induction
of P-A activin expression
by
synaptic
activity
and during
neocortical
development.
Neuroscience
69:781-796.
Bachoo M, Polosa C (1991) Long-term
potentiation
of nicotinic
transmission by a heterosynaptic
mechanism
in the stellate ganglion of the
cat. J Physiol (Lond)
65:639-647.
Bachoo M, Heppner
T, Fiekers
J, Polosa C (1992) A role for protein
kinase C in long-term
potentiation
of nicotinic
transmission
in the
superior cervical ganglion of the rat. Brain Res 585:299-302.
Bailey CH, Montarolo
PG, Chen M, Kandel
ER, Schacher
S (1992)
Inhibitors
of protein
and RNA synthesis block the structural
changes
that accompany
long-term
facilitation
in Aplysiu. Neuron
9:749-758.
Barondes SH (1984) Soluble lectins: a new class of extracellular
proteins.
Science 223:1259-1264.
Barondes SH (1988) Bifunctional
properties
of lectins: lectins redefined.
Trends Biol Sci 13:480-482.
Binder LI, Frankfurter
A, Rebhun
LI (1985) The distribution
of tau in
the mammalian
central nervous system. J Cell Biol 101:1371-1378.
Bliss TVP, Collingridge
GL (1993) A synaptic model of memory:
longterm potentiation
in the hippocampus.
Nature 361:31-39.
Blum AS, Barnstable
CJ (1987) 0acetylation
of a cell-surface
carbohydrate creates discrete molecular
patterns
during neural development.
Proc Nat1 Acad Sci USA 84:8716-8720.
Breviara
F, d’Aniello
EM, Golay J, Peri G, Bottazzi B, Bairoch A, Saccone
S, Marzella
R, Predazzi V, Rocchi M, Valle GD, Dejana E, Mantovani
A, Introna M (1992) Interleukin-l-inducible
genes in endothelial
cells.
J Biol Chem 267:22190-22197.
Chen C, Okayamam
H (1987) High-efficiency
transformation
of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745-2752.
Clerch LB, Whitney
P, Hass M, Drew K, Miller T, Werner
R, Massaro D
(1988) Sequence of a full-length
cDNA for rat lung P-galactosidasebinding protein:
primary
and secondary
structure
of the lectin. Biochemistry
27:692-699.
Cohen C, Parry D (1994) a-helical
coiled coils: more facts and better
predictions.
Science 263:488-489.
Cole A, Abu-Shakra
S, Saffen D, Baraban J, Worley P (1990) Rapid rise
in transcription
factor messenger RNAs in rat brain after electroshock
induced seizures. J Neurochem
55:1920-1927.
Copeland
NG, Jenkins NA (1991) Development
and applications
of a
molecular
genetic linkage map of the mouse genome.
Trends Genet
7:113-118.
Coria F, Castano E, Prelli F, Larrondo-Lillo
M, van Duinen S, Shelanski
ML, Frangione
B (1988) Isolation
and characterization
of amyloid P
component
from Alzheimer’s
disease and other types of cerebral amyloidosis. Lab Invest 58:454-458.
Davis HP, Squire LR (1984) Protein
synthesis and memoty:
a review.
Physiol Bull 96:518-559.
DeGrauw
TJ, Liwnicz
BH (1986) Lectins are markers
of neuronal
migration and differentiation
in rat brain. Dev Neurosci
8:236-242.
Dodd J, Jesse11 TM (1985) Lactoseries
carbohydrates
specify subsets of
dorsal root ganglion neurons projecting
to superficial
dorsal horn of rat
spinal cord. J Neurosci 5:3278-3294.
Dodd J, Solter D, Jesse11 TM (1984) Monoclonal
antibodies
against
carbohydrate
differentiation
antigens identify subsets of primary
sensory
neurons. Nature 311:469-472.
Dowton
SB, McGrew
SD (1990) Rat serum amyloid P component.
Biothem J 270:553-556.
Emsley J, White HE, O’Hara
BP, Oliva G, Srinivasan
N, Tickle
IJ,
Blundell
TL, Pepys MB, Wood SP (1994) Structure
of pentameric
human serum amyloid P component.
Nature 367:338-345.
Flexner JB, Flexner LB, Stellar E (1963) Memory
in mice as affected by
intracerebral
puromycin.
Science 141:57-59.
Fox K, Daw NW (1993) Do NMDA
receptors
have a critical function
in
visual cortical plasticity?
Trends Neurosci
16:116-122.
Frey U, Huang YY, Kandel ER (1993) Effects of CAMP stimulate a late
stage of LTP in hippocampal
CA1 neurons.
Science 260:1661-1664.
Goelet P, Castellucci
V, Schacher S, Kandel E (1986) The long and the
short
of long-term
memory:
a molecular
framework.
Nature
3221419-422.
Green EL (1981) Linkage, recombination
and mapping.
New York: Oxford UP.
Hind CRK, Collins PM, Renn D, Cook RB, Caspi D, Baltz ML, Pepys MB
(1984) Binding specificity of serum amyloid P component
for the pyruvate acetal of galactose. J Exp Med 159:1058-1069.
J. Neurosci.,
April
15, 1996,
16(8):2463-2478
2477
Hsu YC, Perin MS (1995) Human neuronal pentraxin
II (NPTX2):
conservation,
genomic structure,
and chromosomal
localization.
Genomics
28:220-227.
Huettner
JE (1990) Glutamate
receptor
channels in rat DRG neurons:
activation
by kainate and quisqualate
and blockade of desensitization
by
Con A. Neuron
5~255-266.
Hynes MA, Gitt M, Barondes SH, Jesse11 TM, Buck LB (1990) Selective
expression
of an endogenous
lactose-binding
lectin gene in subsets of
central and peripheral
neurons. J Neurosci
10:1004-1013.
Jenkins NA, Copeland
NG, Taylor BA, Lee BK, (1982) Organization,
distribution,
and stability
of endogenous
ecotropic
murine
leukemia
virus DNA
sequences
in chromosomes
of Mus musculuus.
J Virol
43126-36.
Jordan CA (1990) In situ hybridization
in cells and tissue sections: a study
of myelin gene expression
during CNS myelination
and remyelination.
In: In situ hybridization
histochemistry,
pp 39-70.
Boca Raton, FL:
CRC.
Joubert R, Caron M, Bladier D (1987) Brain lectin-mediated
agglutinability of dissociated
cells from embryonic
and postnatal
mouse brain.
Dev Brain Res 36:146-150.
Kaufmann
WE, Worley
PF, Pegg J, Bremer
M, Isakson Cox P (1996)
Cox-2, a synaptically-induced
enzyme, is expressed by excitatory
neurons at postsynaptic
sites in rat cerebral
cortex. Proc Nat1 Acad Sci
USA, in press.
Kehoe J (1978) Transformation
by concanavalin
A of the response of
molluscan
neurons to t-glutamate.
Nature 274:866-869.
Kennedy
TE, Serafini
T, de la Torre JR, Tessier-Lavigne
M (1994)
Netrins are diffusible
chemotropic
factors for commissural
axons in the
embryonic
spinal cord. Cell 78:425-435.
Kobiler
D, Beyer EC, Barondes
SH (1978) Developmentally
regulated
lectins from chick muscle, brain, and liver have similar chemical
and
immunological
properties.
Dev Biol 64:265-272.
Kozak M (1987) At least six nucleotides
preceding
the AUG initiator
codon enhance translation
in mammalian
cells. J Mol Biol 196:947-950.
Kuchler S, Fressinaud
C, Sarlieve LL, Vincendon
G, Zanetta JP (1988)
Cerebellar
soluble lectin is responsible
for cell adhesion and participates in myelin compaction
in cultured rat oligodendrocytes.
Dev Neurosci 10:199-212.
Lau LF, Nathans D (1987) Expression
of a set of growth-related
immediate early genes in BALB/c3T3
cells: coordinate
regulation
with c-fos
or c-myc. Proc Nat1 Acad Sci USA 84:1182-1186.
Lee G, Lee TH, Vicek J (1993) TSG-14,
a tumor necrosis factor- and
IL-l-inducible
protein,
is a novel member
of the pentraxin
family of
acute phase proteins.
J Immunol
150:1804-1812.
Lemmon
V, Burden
SM, Payne HR, Elmslie
GJ, Hlavin
ML (1992)
Neurite
growth
on different
substrates:
permissive
versus instructive
influences
and the role of adhesive strength. J Neurosci
12:818-826.
Lin SS, Levitan
IB (1987) Concanavalin
A alters synaptic
specificity
between cultured Aplysia neurons. Science 237:648-650.
Lin SS, Levitan IB (1991) Concanavalin
A: a tool to investigate
neuronal
plasticity.
Trends Neurosci
14:273-277.
Linzer DIH, Nathans D (1983) Growth-related
changes in specific mRNAs
of cultured mouse cells. Proc Nat1 Acad Sci USA 80:4271-4275.
Lyford G, Yamagata
K, Kaufmann
WE, Barnes CA, Sanders LK, Copeland NG, Gilbert DJ, Jenkins NA, Lanahan AA, Worley PF (1995) Arc,
a growth
factor
and activity-regulated
gene
encodes
a novel
cytoskeleton-associated
protein that is enriched in neuronal
dendrites.
Neuron
14:433-445.
Miller KD, Chapman
B, Stryker MP (1989) Visual responses in adult cat
visual cortex depend
on N-methyl-D-aspartate
receptors.
Proc Natl
Acad Sci USA 86:5183-5187.
Montarolo
PG, Goelet P, Castellucci
V, Morgan
J, Kandel E, Schacher S
(1986) A critical
period for macromolecular
synthesis in long-term
heterosynaptic
facilitation
in Aplysia. Science 234:1249-1254.
Nedivi E, Hevroni
D, Naot D, Israeli D, Citri Y (1993) Numerous
candidate plasticity-related
genes revealed
by differential
cDNA cloning.
Nature 363:713-722.
Nguyen PV, Abel T, Kandel ER (1994) Requirement
of a critical period
of transcription
for induction
of a late phase of LTP. Science
265:1104-1106.
Noland TD, Friday BB, Maulit MT, Gerton GL (1994) The sperm acrosomal matrix
contains
a novel member
of the pentraxin
family of
calcium-dependent
binding proteins.
J Biol Chem 269:32607-32614.
Patterson
SL, Grover
LM, Schwartzkroin
PA, Bothwell
M (1992) Neurotrophin
expression
in rat hippocampal
slices: a stimulus
paradigm
2478
J. Neurosci.,
April
15, 1996,
76(8):2463-2478
inducing LTP in CA1 evokes increases in BDNF
and NT-3 messengerRNAs. Neuron 9:1081-1088.
Pennypacker
K, Fischer I, Levitt P (1991) Early in vitro genesis and
differentiation
of axons and dendrites
by hippocampal
neurons analyzed
quantitatively
with neurofilament-H
and microtubule-associated
protein
2 antibodies.
Exp Neurol 111:25.
Pepys MB, Rademacher
TW, Amatayakul-Chantler
S, Williams
P, Nobel
GE, Hutchinson
WL, Hawkins
PN, Nelson SR, Gallimore
JR, Herbert
J, Hutton T, Dwek RA (1994) Human serum amyloid P component
is
an invariant
constituent
of amyloid deposits and has a uniquely
homogeneous glycostructure.
Proc Nat1 Acad Sci USA 91:5602-5606.
Pongor S, Hatsagi Z, Degtyarenko
K, Fabian P, Skerl V, Hegyi H, Murvai
J, Bevilacqua
V (1994) The SBASE protein
domain
library,
release
3.0: a collection of annotated
protein sequence segments. Nucleic Acids
Res 22:3610-3615.
Qian Z, Gilbert
ME, Colicos MA, Kandel ER, Kuhl D (1993) Tissueplasminogen
activator
is induced as an immediate-early
gene during
seizure, kindling and long-term
potentiation.
Nature
361:453-457.
Rassouli M, Sambasivam
H, Azadis P, Dell A, Morris HR, Nagpurkar
A,
Mookerjea
S, Murray
RK (1992) Derivation
of the amino acid sequence of rat C-reactive
protein
from cDNA cloning with additional
studies
on the nature
of its dimeric
component.
J Biol Chem
267~2947-2954.
Regan LJ, Dodd J, Barondes SH, Jesse11 TM (1986) Selective expression
of endogenous
lactose-binding
lectins and lactoseries
glycoconjugates
in
subsets of rat sensory neurons. Proc Nat1 Acad Sci USA 83:2248-2252.
Reid M, Blobel CP (1994) Apexin, an acrosomal
pentaxin.
J Biol Chem
269:32615-32620.
Rinaman
L, Card JP, Enquist LW (1993) Spatiotemporal
responses of
astrocytes,
ramified
microglia,
and brain macrophages
to central neuronal infection
with pseudorabies
virus. J Neurosci
13:685-720.
Rost B, Sander C (1993) Prediction
of protein
structure
at better than
70% accuracy. J Mol Biol 232:584-599.
Rost B, Sander C (1994) Combining
evolutionary
information
and neural
networks
to predict protein secondary
structure.
Proteins 19:55-72.
Saffen DW, Cole AJ, Worley
PF, Christy
BA, Ryder
K, Baraban
JM
(1988) Convulsant-induced
increase in transcription
factor messenger
RNAs in rat brain. Proc Nat1 Acad Sci USA 85:7795-7799.
Schlimgen
AK, Helms JA, Vogel H, Perin MS (1995) Neuronal
pentraxin, a secreted protein with homology
to acute phase proteins of the
immune system. Neuron
14:519-526.
Schwalbe RA, Dahlback
B, Coe JE, Nelsestuen
GL (1992) Pentraxin
family of proteins
interact
specifically
with phosphorylcholine
and/or
phosphotylethanolamine.
Biochemistry
31:4907-4915.
Shatz C (1990) Impulse activity and the patterning
of connections
during
CNS development.
Neuron
5:745-756.
Shaw G, Kamen R (1986) A conserved
AU sequence from the 3’ untranslated
region of GM-CSF
mRNA
mediates selective mRNA
degredation.
Cell 46:659-667.
Sheng M, Greenberg
M (1990) The regulation
and function of c-fos and
other immediate
early genes in the nervous system. Neuron 4:477-485.
Shinozaki
H, Ishida M (1979) Pharmacological
distinction
between the
excitatory
junctional
potential
and the glutamate
potential
revealed by
concanavalin
A at the crayfish
neuromuscular
junction.
Brain Res
161:493-501.
Tsui et al.
l
Narp
Expression
and
Regulation
in Brain
Siegel J, Rent R, Gewurz
H (1974) Interactions
of C-reactive
protein
with the complement.
J Exp Med 140:631-647.
Siegel J, Osmand AP, Wilson MR (1975) Interaction
of C-reactive
protein with the complement
system. J Exp Med 142:709-721.
Silva AJ, Giese PK (1994) Plastic genes are in! Curr Opin Neurobiol
4:413-420.
Simpson DL, Thorne
DR, Loh HH (1977) Developmentally
regulated
lectin in neonatal
rat brain. Nature 266:367-369.
Singh G, Kaur S, Stock JL, Jenkins NA, Gilbert DJ, Copeland
NG, Potter
SS (1991) Identification
of 10 murine
homeobox
genes. Proc Nat1
Acad Sci USA 88:10706-10710.
Sisodia SS, Koo EH, Beyreuther
K, Unterbeck
A, Price D (1990) Evidence that beta-amyloid
protein in Alzheimer’s
disease in not derived
by normal processing.
Science 248:492-495.
Slunt HH, TG, Von Koch C, Lo A, Tanzi RE, Sisodia SS (1994) Expression of a ubiquitous,
cross-reactive
homologue
of the mouse fi-amyloid
precursor
protein
(APP). J Biol Chem 269:2637-2644.
Thio LL, Clifford DB, Zorumski
CF (1992) Blockade of ionotropic
quisqualate receptor desensitization
in rat hippocampal
neurons by wheatgerm agglutinin
and other lectins. J Neurol Sci 52:35-44.
Thio LL, Clifford DB, Zorumski
CF (1993) Blockade of ionotropic
quisqualate receptor
desensitization
in rat hippocampal
neurons by wheatgerm agglutinin
and other lectins. Neuroscience
52:35-44.
von Heijne G (1986) A new method for predicting
signal sequence cleavage sites. Nucleic Acids Res 14:4683-4690.
Waters RS, McCandlish
CA, Cooper NG (1990) Early development
of Sl
cortical barrel subfield representation
of forclimb
in normal and deafferented
neonatal
rat as delineated
by peroxidasc
conjugated
lcctin,
peanut agglutinin
(PNA).
Exp Brain Res 81:234-240.
Worley PF, Cole AJ, Murphy
TM, Christy BA, Nakabeppu
Y, Baraban JM
(1990) Synaptic
regulation
of immediate
early genes in brain. Cold
Spring Harb Symp Quant Biol 55:213-223.
Worley PF, Cole AJ, Murphy
TM, Christy BA, Nakabeppu
Y, Baraban JM
(1991) Constitutive
expression
of ~$68
in neocortex
is regulated
by
synaptic activity. Proc Nat1 Acad Sci USA 88:5106-5110.
Worley
PF, Bhat RV, Baraban
JM, Erickson
CA, McNaughton
BL,
Barnes CA (1993) Thresholds
for synaptic activation
of transcription
factors
in hippocampus:
correlation
with long-term
enhancement.
J Neurosci
13:4776-4786.
Yamagata K Andreasson
KI, Kaufmann
WE, Barnes CA, Worley PF (1993)
Expression
of a mitogen-inducible
cyclooxygenase
in brain neurons: regulation by synaptic activity and glucocorticoids.
Neuron 11:371-386.
Yamagata
K, Kaufmann
WE, Lanahan
A, Papapavlou
M, Barnes CA,
Worley PF (1994a) Egr3/Pilot,
a zinc-finger
transcription
factor, is rapidly regulated
by activity in brain neurons and co-localizes
with Egrl/
Zif268. Learn Memory
1:140-152.
Yamagata
K, Sanders LK, Kaufmann
WE, Barnes CA, Nathans D, Worley
PF (1994b)
Rheb, a growth factor and synaptic activity regulated
gene,
encodes a novel Ras-related
protein. J Biol Chem 269:16333-16339.
Zilles K, Wree A, Schleicher
A, Divac I (1984) The monocular
and
binocular
subfields
of the rat’s primary
visual cortex:
a quantitative
morphological
approach.
J Comp Neurol 226:391-402.